T cell factor 1 (TCF-1) defines T cell differentiation in colorectal cancer.

The presence of precursor to exhausted (Tpex) CD8+ T cells is important to maintain robust immunity following treatment with immune checkpoint inhibition (ICI). Impressive responses to ICI are emerging in patients with stage II-III mismatch repair (MMR)-deficient (dMMR) colorectal cancer (CRC). We found 64% of dMMR and 15% of mismatch repair-proficient (pMMR) stage III CRCs had a high frequency of tumor infiltrating lymphocytes (TIL-hi). Furthermore, expression of TCF-1 (Tcf7) by CD8+ T cells predicted improved patient prognosis and Tpex cells (CD3+CD8+TCF-1+PD-1+) were abundant within lymphoid aggregates of stage III CRCs. In contrast, CD3+CD8+TCF-1-PD-1+ cells were more abundant at the invasive front and tumor core, while γδ T cells were equally abundant in all tumor areas. Interestingly, no differences in the frequency of Tpex cells were observed between TIL-hi dMMR and TIL-hi pMMR CRCs. Therefore, Tpex cell function and ICI response rates in TIL-hi CRC warrants further investigation.

Orthotopic MC-38 Allograft as a Robust Preclinical Model of Colorectal Carcinoma.

Colorectal cancer (CRC) is a significant global health concern, requiring effective preclinical models for studying its development and testing therapies. Mouse models have been used, including spontaneous tumors, carcinogen exposure, and tumor cell implantation as xenografts or at orthotopic sites. Here, we describe an orthotopic preclinical model of CRC, which provides a valuable tool for studying tumor growth and the tumor microenvironment, offering a more accurate representation of human CRC compared to xenograft models.

Hostile bile limits anti-cancer immunity.

Various microbial metabolites promote cell transformation. In this issue of Immunity, Cong et al. show that deoxycholic acid (DCA), a microbial metabolite of bile, promotes tumor growth by suppressing antitumor CD8+ T cell responses via dysregulation of calcium efflux.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

Divergent molecular networks program functionally distinct CD8+ skin-resident memory T cells.

Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

cellCounts: an R function for quantifying 10x Chromium single-cell RNA sequencing data.

SUMMARY: The 10x Genomics Chromium single-cell RNA sequencing technology is a powerful gene expression profiling platform, which is capable of profiling expression of thousands of genes in tens of thousands of cells simultaneously. This platform can produce hundreds of million reads in a single experiment, making it a very challenging task to quantify expression of genes in individual cells due to the massive data volume. Here, we present cellCounts, a new tool for efficient and accurate quantification of Chromium data. cellCounts employs the seed-and-vote strategy to align reads to a reference genome, collapses reads to Unique Molecular Identifiers (UMIs) and then assigns UMIs to genes based on the featureCounts program. Using both simulation and real datasets for evaluation, cellCounts was found to compare favourably to cellRanger and STARsolo. cellCounts is implemented in R, making it easily integrated with other R programs for analysing Chromium data. AVAILABILITY AND IMPLEMENTATION: cellCounts was implemented as a function in R package Rsubread that can be downloaded from http://bioconductor.org/packages/release/bioc/html/Rsubread.html. Data and analysis code used in this study can be freely accessed via La Trobe University's Institutional Repository at https://doi.org/10.26181/21588276.

Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression.

Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.

Dendritic cells shape TCF1+CD8+ progenitor T cell heterogeneity.

In two elegant studies, Tyler Jacks' group and colleagues unveil crucial interactions between dendritic cells and TCF1+CD8+ progenitor T cells, shaping their heterogeneity and offering potential to design new putative cancer immunotherapies and vaccines.

Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.

Type 2 Innate Lymphoid Cells Protect against Colorectal Cancer Progression and Predict Improved Patient Survival.

Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.

Loss of NFKB1 Results in Expression of Tumor Necrosis Factor and Activation of Signal Transducer and Activator of Transcription 1 to Promote Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Activity of nuclear factor κB transcription factors and signaling via signal transducer and activator of transcription (STAT) are frequently altered in gastric cancer cells. Mice lacking NFKB1 (Nfkb1-/- mice) develop invasive gastric cancer, and their gastric tissues have increased levels of cytokines, such as interleukin (IL) 6, IL22, IL11, and tumor necrosis factor (TNF), as well as increased activation of STAT1. We investigated whether these cytokines were required for STAT1 activation in gastric tissues of mice and critical for gastric tumorigenesis. METHODS: We crossed Nfkb1-/- mice with Il6-/-, Il22-/-, Il11Rα-/-, and Tnf-/- mice. Stomach tissues from compound mutant mice were analyzed by histology, immunoblotting, and RNA sequencing. Lymphoid, myeloid, and epithelial cells were isolated from stomachs, and the levels of cytokines were determined by flow cytometric analysis. RESULTS: Nfkb1-/- mice developed gastritis, oxyntic atrophy, gastric dysplasia, and invasive tumors, whereas Nfkb1-/-Stat1-/- mice did not, even when followed for as long as 2 years. The levels of Il6, Il11, Il22, and Tnf messenger RNA were increased in the body and antrum of the stomachs from Nfkb1-/- mice, from 3-6 months of age. However, Nfkb1-/-Il6-/-, Nfkb1-/-Il22-/-, and Nfkb1-/-Il11Rα-/- mice still developed gastric tumors, although the absence of IL11 receptor (IL11R) significantly reduced development of invasive gastric tumors. Stomachs from Nfkb1-/-Tnf-/- mice exhibited significantly less gastritis and oxyntic atrophy and fewer tumors than Nfkb1-/- mice. This correlated with reduced activation of STAT1 and STAT3 and fewer numbers of T cells and B cells infiltrating the gastric body. Loss of STAT1 or TNF significantly reduced expression of PD-L1 on epithelial and myeloid (CD11b+) cells in the gastric mucosa of Nfkb1-/- mice-indeed, to the levels observed on the corresponding cells from wild-type mice. CONCLUSIONS: In studies of gastric tumor development in knockout mice, we found that loss of NFKB1 causes increased expression of TNF in the stomach and thereby drives activation of STAT1, resulting in an inflammatory immune response and the development of gastric cancer. IL11R appears to be required for the progression of gastric tumors to the invasive stage. These findings suggest that inhibitors of TNF, and possibly also inhibitors of IL11/IL11Rα, might be useful in the treatment of gastric cancer.

Control of Lymphocyte Fate, Infection, and Tumor Immunity by TCF-1.

T cell factor-1 (TCF-1), encoded by Tcf7, is a transcription factor and histone deacetylase (HDAC) essential for commitment to both the T cell and the innate lymphoid cell (ILC) lineages in mammals. In this review, we discuss the multifunctional role of TCF-1 in establishing these lineages and the requirement for TCF-1 throughout lineage differentiation and maintenance of lineage stability. We highlight recent reports showing promise for TCF-1 as a novel biomarker to identify recently characterized subsets of exhausted CD8+ T cells that may help to predict patient responses to immune checkpoint blockade (ICB).

TCF-1 limits the formation of Tc17 cells via repression of the MAF-RORγt axis.

Interleukin (IL)-17-producing CD8+ T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ-producing effector CD8+ T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 (Tcf7) regulates CD8+ T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1-driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8+ T cell subsets. IL-17-producing CD8+ T cells isolated from healthy humans were also distinct from CD8+IL-17- T cells and enriched in pathways driven by MAF and RORγt Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT.

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.

NLRP1 restricts butyrate producing commensals to exacerbate inflammatory bowel disease.

Anti-microbial signaling pathways are normally triggered by innate immune receptors when detecting pathogenic microbes to provide protective immunity. Here we show that the inflammasome sensor Nlrp1 aggravates DSS-induced experimental mouse colitis by limiting beneficial, butyrate-producing Clostridiales in the gut. The colitis-protective effects of Nlrp1 deficiency are thus reversed by vancomycin treatment, but recapitulated with butyrate supplementation in wild-type mice. Moreover, an activating mutation in Nlrp1a increases IL-18 and IFNγ production, and decreases colonic butyrate to exacerbate colitis. We also show that, in patients with ulcerative colitis, increased NLRP1 in inflamed regions of the colon is associated with increased IFN-γ. In this context, NLRP1, IL-18 or IFN-γ expression negatively correlates with the abundance of Clostridiales in human rectal mucosal biopsies. Our data identify the NLRP1 inflammasome to be a key negative regulator of protective, butyrate-producing commensals, which therefore promotes inflammatory bowel disease.

Plasmacytoid dendritic cell heterogeneity is defined by CXCL10 expression following TLR7 stimulation.

Plasmacytoid dendritic cells (pDCs) play a critical role in bridging the innate and adaptive immune systems. pDCs are specialized type I interferon (IFN) producers, which has implicated them as initiators of autoimmune pathogenesis. However, little is known about the downstream effectors of type I IFN signaling that amplify autoimmune responses. Here, we have used a chemokine reporter mouse to determine the CXCR3 ligand responses in DCs subsets. Following TLR7 stimulation, conventional type 1 and type 2 DCs (cDC1 and cDC2, respectively) uniformly upregulate CXCL10. By contrast, the proportion of chemokine positive pDCs was significantly less, and stable CXCL10+ and CXCL10- populations could be distinguished. CXCL9 expression was induced in all cDC1s, in half of the cDC2 but not by pDCs. The requirement for IFNAR signaling for chemokine reporter expression was interrogated by receptor blocking and deficiency and shown to be critical for CXCR3 ligand expression in Flt3-ligand-derived DCs. Chemokine-producing potential was not concordant with the previously identified markers of pDC heterogeneity. Finally, we show that CXCL10+ and CXCL10- populations are transcriptionally distinct, expressing unique transcriptional regulators, IFN signaling molecules, chemokines, cytokines, and cell surface markers. This work highlights CXCL10 as a downstream effector of type I IFN signaling and suggests a division of labor in pDCs subtypes that likely impacts their function as effectors of viral responses and as drivers of inflammation.

Characterization of Blimp-1 function in effector regulatory T cells.

Regulatory T (Treg) cells maintain immunological tolerance in steady-state and after immune challenge. Activated Treg cells can undergo further differentiation into an effector state that highly express genes critical for Treg cell function, including ICOS, TIGIT and IL-10, although how this process is controlled is poorly understood. Effector Treg cells also specifically express the transcriptional regulator Blimp-1 whose expression overlaps with many of the canonical markers associated with effector Treg cells, although not all ICOS+TIGIT+ Treg cells express Blimp-1 or IL-10. In this study, we addressed the role of Blimp-1 in effector Treg cell function. Mice lacking Blimp-1 specifically in Treg cells mature normally, but succumb to a multi-organ inflammatory disease later in life. Blimp-1 is not required for Treg cell differentiation, with mutant mice having increased numbers of effector Treg cells, but regulated a suite of genes involved in cell signaling, communication and survival, as well as being essential for the expression of the immune modulatory cytokine IL-10. Thus, Blimp-1 is a marker of effector Treg cells in all contexts examined and is required for the full functionality of these cells during aging.

Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner.

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.

The TNF Receptor Superfamily-NF-κB Axis Is Critical to Maintain Effector Regulatory T Cells in Lymphoid and Non-lymphoid Tissues.

After exiting the thymus, Foxp3+ regulatory T (Treg) cells undergo further differentiation in the periphery, resulting in the generation of mature, fully suppressive effector (e)Treg cells in a process dependent on TCR signaling and the transcription factor IRF4. Here, we show that tumor necrosis factor receptor superfamily (TNFRSF) signaling plays a crucial role in the development and maintenance of eTreg cells. TNFRSF signaling activated the NF-κB transcription factor RelA, which was required to maintain eTreg cells in lymphoid and non-lymphoid tissues, including RORγt+ Treg cells in the small intestine. In response to TNFRSF signaling, RelA regulated basic cellular processes, including cell survival and proliferation, but was dispensable for IRF4 expression or DNA binding, indicating that both pathways operated independently. Importantly, mutations in the RelA binding partner NF-κB1 compromised eTreg cells in humans, suggesting that the TNFRSF-NF-κB axis was required in a non-redundant manner to maintain eTreg cells in mice and humans.