FOXO1 is a master regulator of memory programming in CAR T cells.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

Transcript-specific enrichment enables profiling rare cell states via scRNA-seq.

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

Convergent epigenetic evolution drives relapse in acute myeloid leukemia.

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.

Acute myeloid leukemia (or AML for short) is a type of blood cancer characterized by abnormally high production of immature white blood cells. Despite advances in AML treatment, many patients relapse after an initially successful first round of treatment. As a result, understanding the factors contributing to relapse is essential for developing effective treatments for the disease. Like most cancers, AML can evolve because of changes to the DNA sequence in cells that cause them to grow uncontrollably or resist treatment. Alongside these genetic mutations, AML cells also undergo 'epigenetic' changes, where regions of the DNA are modified and genes can be switched on or off without altering the DNA sequence. Previous research has demonstrated that epigenetic changes contribute to the development of AML, however, it was not clear if these changes could also make cells resistant to treatment without acquiring new DNA mutations. Nuno, Azizi et al. addressed this question by analyzing the epigenetic states of AML cells from 26 patients at the time of their diagnosis and after treatment when the disease had relapsed. Analysis revealed that almost half of the patients with AML experienced a relapse without acquiring new DNA mutations. Instead, these AML cells developed specific epigenetic changes that helped them to resist cancer treatment. Moreover, studying individual AML cells from different patients showed that the cells became more epigenetically similar at relapse, suggesting that they converge towards a more treatment-resistant disease. Future experiments will determine exactly how these epigenetic changes lead to treatment resistance. Currently, most of the drugs used to treat AML are either chemotherapies or ones that target specific DNA mutations. The findings of Nuno, Azizi et al. suggest that drugs targeting specific epigenetic changes may be more effective for some patients. Further studies will be needed to determine which patients may benefit and which epigenetic drugs could be useful.

Regulation of immune signal integration and memory by inflammation-induced chromosome conformation.

3-dimensional (3D) genome conformation is central to gene expression regulation, yet our understanding of its contribution to rapid transcriptional responses, signal integration, and memory in immune cells is limited. Here, we study the molecular regulation of the inflammatory response in primary macrophages using integrated transcriptomic, epigenomic, and chromosome conformation data, including base pair-resolution Micro-Capture C. We demonstrate that interleukin-4 (IL-4) primes the inflammatory response in macrophages by stably rewiring 3D genome conformation, juxtaposing endotoxin-, interferon-gamma-, and dexamethasone-responsive enhancers in close proximity to their cognate gene promoters. CRISPR-based perturbations of enhancer-promoter contacts or CCCTC-binding factor (CTCF) boundary elements demonstrated that IL-4-driven conformation changes are indispensable for enhanced and synergistic endotoxin-induced transcriptional responses, as well as transcriptional memory following stimulus removal. Moreover, transcriptional memory mediated by changes in chromosome conformation often occurred in the absence of changes in chromatin accessibility or histone modifications. Collectively, these findings demonstrate that rapid and memory transcriptional responses to immunological stimuli are encoded in the 3D genome.

Inosine induces stemness features in CAR-T cells and enhances potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR-T cells express CD39 and CD73, which mediate proximal steps in Ado generation. Here, we sought to enhance CAR-T cell potency by knocking out CD39, CD73, or adenosine receptor 2a (A2aR) but observed only modest effects. In contrast, overexpression of Ado deaminase (ADA-OE), which metabolizes Ado to inosine (INO), induced stemness and enhanced CAR-T functionality. Similarly, CAR-T cell exposure to INO augmented function and induced features of stemness. INO induced profound metabolic reprogramming, diminishing glycolysis, increasing mitochondrial and glycolytic capacity, glutaminolysis and polyamine synthesis, and reprogrammed the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR-T cell products meeting criteria for clinical dosing. These results identify INO as a potent modulator of CAR-T cell metabolism and epigenetic stemness programming and deliver an enhanced potency platform for cell manufacturing.

Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion.

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not-rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non-rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.

FOXO1 is a master regulator of CAR T memory programming.

Poor CAR T persistence limits CAR T cell therapies for B cell malignancies and solid tumors1,2. The expression of memory-associated genes such as TCF7 (protein name TCF1) is linked to response and long-term persistence in patients3-7, thereby implicating memory programs in therapeutic efficacy. Here, we demonstrate that the pioneer transcription factor, FOXO1, is responsible for promoting memory programs and restraining exhaustion in human CAR T cells. Pharmacologic inhibition or gene editing of endogenous FOXO1 in human CAR T cells diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype, and impaired antitumor activity in vitro and in vivo. FOXO1 overexpression induced a gene expression program consistent with T cell memory and increased chromatin accessibility at FOXO1 binding motifs. FOXO1-overexpressing cells retained function, memory potential, and metabolic fitness during settings of chronic stimulation and exhibited enhanced persistence and antitumor activity in vivo. In contrast, TCF1 overexpression failed to enforce canonical memory programs or enhance CAR T cell potency. Importantly, endogenous FOXO1 activity correlated with CAR T and TIL responses in patients, underscoring its clinical relevance in cancer immunotherapy. Our results demonstrate that memory reprogramming through FOXO1 can enhance the persistence and potency of human CAR T cells and highlights the utility of pioneer factors, which bind condensed chromatin and induce local epigenetic remodeling, for optimizing therapeutic T cell states.

Latent human herpesvirus 6 is reactivated in CAR T cells.

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

Convergent Epigenetic Evolution Drives Relapse in Acute Myeloid Leukemia.

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

Single-cell multi-omics of mitochondrial DNA disorders reveals dynamics of purifying selection across human immune cells.

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

Clonal hematopoiesis is associated with protection from Alzheimer's disease.

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant expansion of mutated hematopoietic stem cells. As CHIP-associated mutations are known to alter the development and function of myeloid cells, we hypothesized that CHIP may also be associated with the risk of Alzheimer's disease (AD), a disease in which brain-resident myeloid cells are thought to have a major role. To perform association tests between CHIP and AD dementia, we analyzed blood DNA sequencing data from 1,362 individuals with AD and 4,368 individuals without AD. Individuals with CHIP had a lower risk of AD dementia (meta-analysis odds ratio (OR) = 0.64, P = 3.8 × 10-5), and Mendelian randomization analyses supported a potential causal association. We observed that the same mutations found in blood were also detected in microglia-enriched fraction of the brain in seven of eight CHIP carriers. Single-nucleus chromatin accessibility profiling of brain-derived nuclei in six CHIP carriers revealed that the mutated cells comprised a large proportion of the microglial pool in the samples examined. While additional studies are required to validate the mechanistic findings, these results suggest that CHIP may have a role in attenuating the risk of AD.

Inosine Induces Stemness Features in CAR T cells and Enhances Potency.

Adenosine (Ado) mediates immune suppression in the tumor microenvironment and exhausted CD8+ CAR T cells mediate Ado-induced immunosuppression through CD39/73-dependent Ado production. Knockout of CD39, CD73 or A2aR had modest effects on exhausted CAR T cells, whereas overexpression of Ado deaminase (ADA), which metabolizes Ado to inosine (INO), induced stemness features and potently enhanced functionality. Similarly, and to a greater extent, exposure of CAR T cells to INO augmented CAR T cell function and induced hallmark features of T cell stemness. INO induced a profound metabolic reprogramming, diminishing glycolysis and increasing oxidative phosphorylation, glutaminolysis and polyamine synthesis, and modulated the epigenome toward greater stemness. Clinical scale manufacturing using INO generated enhanced potency CAR T cell products meeting criteria for clinical dosing. These data identify INO as a potent modulator of T cell metabolism and epigenetic stemness programming and deliver a new enhanced potency platform for immune cell manufacturing.

Skin basal cell carcinomas assemble a pro-tumorigenic spatially organized and self-propagating Trem2+ myeloid niche.

Cancer immunotherapies have revolutionized treatment but have shown limited success as single-agent therapies highlighting the need to understand the origin, assembly, and dynamics of heterogeneous tumor immune niches. Here, we use single-cell and imaging-based spatial analysis to elucidate three microenvironmental neighborhoods surrounding the heterogeneous basal cell carcinoma tumor epithelia. Within the highly proliferative neighborhood, we find that TREM2+ skin cancer-associated macrophages (SCAMs) support the proliferation of a distinct tumor epithelial population through an immunosuppression-independent manner via oncostatin-M/JAK-STAT3 signaling. SCAMs represent a unique tumor-specific TREM2+ population defined by VCAM1 surface expression that is not found in normal homeostatic skin or during wound healing. Furthermore, SCAMs actively proliferate and self-propagate through multiple serial tumor passages, indicating long-term potential. The tumor rapidly drives SCAM differentiation, with intratumoral injections sufficient to instruct naive bone marrow-derived monocytes to polarize within days. This work provides mechanistic insights into direct tumor-immune niche dynamics independent of immunosuppression, providing the basis for potential combination tumor therapies.

The temporal progression of immune remodeling during metastasis.

Tumor metastasis requires systemic remodeling of distant organ microenvironments which impacts immune cell phenotypes, population structure, and intercellular communication networks. However, our understanding of immune phenotypic dynamics in the metastatic niche remains incomplete. Here, we longitudinally assayed lung immune cell gene expression profiles in mice bearing PyMT-driven metastatic breast tumors from the onset of primary tumorigenesis, through formation of the pre-metastatic niche, to the final stages of metastatic outgrowth. Computational analysis of these data revealed an ordered series of immunological changes that correspond to metastatic progression. Specifically, we uncovered a TLR-NFκB myeloid inflammatory program which correlates with pre-metastatic niche formation and mirrors described signatures of CD14+ 'activated' MDSCs in the primary tumor. Moreover, we observed that cytotoxic NK cell proportions increased over time which illustrates how the PyMT lung metastatic niche is both inflammatory and immunosuppressive. Finally, we predicted metastasis-associated immune intercellular signaling interactions involving Igf1 and Ccl6 which may organize the metastatic niche. In summary, this work identifies novel immunological signatures of metastasis and discovers new details about established mechanisms that drive metastatic progression. In brief: McGinnis et al. report a longitudinal scRNA-seq atlas of lung immune cells in mice bearing PyMT-driven metastatic breast tumors and identify immune cell transcriptional states, shifts in population structure, and rewiring of cell-cell signaling networks which correlate with metastatic progression. Highlights: Longitudinal scRNA-seq reveals distinct stages of immune remodeling before, during, and after metastatic colonization in the lungs of PyMT mice.TLR-NFκB inflammation correlates with pre-metastatic niche formation and involves both tissue-resident and bone marrow-derived myeloid cell populations. Inflammatory lung myeloid cells mirror 'activated' primary tumor MDSCs, suggesting that primary tumor-derived cues induce Cd14 expression and TLR-NFκB inflammation in the lung. Lymphocytes contribute to the inflammatory and immunosuppressive lung metastatic microenvironment, highlighted by enrichment of cytotoxic NK cells in the lung over time. Cell-cell signaling network modeling predicts cell type-specific Ccl6 regulation and IGF1-IGF1R signaling between neutrophils and interstitial macrophages.

Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.

Cis interactions in the Irf8 locus regulate stage-dependent enhancer activation.

Individual elements within a superenhancer can act in a cooperative or temporal manner, but the underlying mechanisms remain obscure. We recently identified an Irf8 superenhancer, within which different elements act at distinct stages of type 1 classical dendritic cell (cDC1) development. The +41-kb Irf8 enhancer is required for pre-cDC1 specification, while the +32-kb Irf8 enhancer acts to support subsequent cDC1 maturation. Here, we found that compound heterozygous Δ32/Δ41 mice, lacking the +32- and +41-kb enhancers on different chromosomes, show normal pre-cDC1 specification but, surprisingly, completely lack mature cDC1 development, suggesting cis dependence of the +32-kb enhancer on the +41-kb enhancer. Transcription of the +32-kb Irf8 enhancer-associated long noncoding RNA (lncRNA) Gm39266 is also dependent on the +41-kb enhancer. However, cDC1 development in mice remained intact when Gm39266 transcripts were eliminated by CRISPR/Cas9-mediated deletion of lncRNA promoters and when transcription across the +32-kb enhancer was blocked by premature polyadenylation. We showed that chromatin accessibility and BATF3 binding at the +32-kb enhancer were dependent on a functional +41-kb enhancer located in cis Thus, the +41-kb Irf8 enhancer controls the subsequent activation of the +32-kb Irf8 enhancer in a manner that is independent of associated lncRNA transcription.

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells.

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells, 1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.