The CD8 immgenT framework as a universal reference of mouse CD8αβ T cell differentiation states

PD-1 blockade unleashes CD8 T cells, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunctionprograms in tumour-infltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We found both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TILs express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signaling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

Tissue-resident memory T cells (TRM cells) provide protective immunity, but the contributions of specific tissue environments to TRM cell differentiation and homeostasis are not well understood. In the present study, the diversity of gene expression and genome accessibility by mouse CD8+ TRM cells from distinct organs that responded to viral infection revealed both shared and tissue-specific transcriptional and epigenetic signatures. TRM cells in the intestine and salivary glands expressed transforming growth factor (TGF)-β-induced genes and were maintained by ongoing TGF-β signaling, whereas those in the fat, kidney and liver were not. Constructing transcriptional-regulatory networks identified the transcriptional repressor Hic1 as a critical regulator of TRM cell differentiation in the small intestine and showed that Hic1 overexpression enhanced TRM cell differentiation and protection from infection. Provision of a framework for understanding how CD8+ TRM cells adapt to distinct tissue environments, and identification of tissue-specific transcriptional regulators mediating these adaptations, inform strategies to boost protective memory responses at sites most vulnerable to infection.

Resistance to butyrate-induced cell differentiation and apoptosis during spontaneous Caco-2 cell differentiation

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.

Despite its importance in modulating HSV-2 pathogenesis, the nature of tissue-resident immune memory to HSV-2 is not completely understood. We used genital HSV-2 infection of guinea pigs to assess the type and location of HSV-specific memory cells at peripheral sites of HSV-2 infection. HSV-specific antibody-secreting cells were readily detected in the spleen, bone marrow, vagina/cervix, lumbosacral sensory ganglia, and spinal cord of previously-infected animals. Memory B cells were detected primarily in the spleen and to a lesser extent in bone marrow but not in the genital tract or neural tissues suggesting that the HSV-specific antibody-secreting cells present at peripheral sites of HSV-2 infection represented persisting populations of plasma cells. The antibody produced by these cells isolated from neural tissues of infected animals was functionally relevant and included antibodies specific for HSV-2 glycoproteins and HSV-2 neutralizing antibodies. A vigorous IFN-γ-secreting T cell response developed in the spleen as well as the sites of HSV-2 infection in the genital tract, lumbosacral ganglia and spinal cord following acute HSV-2 infection. Additionally, populations of HSV-specific tissue-resident memory T cells were maintained at these sites and were readily detected up to 150 days post HSV-2 infection. Unlike the persisting plasma cells, HSV-specific memory T cells were also detected in uterine tissue and cervicothoracic region of the spinal cord and at low levels in the cervicothoracic ganglia. Both HSV-specific CD4+ and CD8+ resident memory cell subsets were maintained long-term in the genital tract and sensory ganglia/spinal cord following HSV-2 infection. Together these data demonstrate the long-term maintenance of both humoral and cellular arms of the adaptive immune response at the sites of HSV-2 latency and virus shedding and highlight the utility of the guinea pig infection model to investigate tissue-resident memory in the setting of HSV-2 latency and spontaneous reactivation.

During infections, naive CD8 T cells differentiate into terminal effector cells (TE) and long-lived memory CD8 T cells that recirculate through lymphoid organs using the vasculature (TCIRC), and tissue resident memory (TRM) cells that do not recirculate. The transcriptional control that delineates differentiation of TE cells from memory cell subsets is still unclear. DNA motifs recognized by ETS-family transcription factors (TFs) are highly frequent in cis-regulatory regions that become chromatin accessible in naïve CD8 T cells 24 hours after TCR stimulation, and in mature memory T cell subsets. The ETS-family is encoded by 27 genes, and expression analyses revealed Ets1 is the most highly expressed member. Ets1 is transiently downregulated during activation and is re-expressed in TCIRC cells, but remains lower in intestinal TRM cells. P14 CD8 T cells responding to acute LCMV infection that were depleted of Ets1 using retrovirally-delivered shRNAmirs preferentially became KLRG1hi CD127lo TE-like cells, and their numbers in the circulation rapidly declined, resulting in an absence of long-lived effector cells and residual numbers of central memory-like cells. Concomitantly, Ets1-depleted cells re-distributed into the gut TRM compartment and had increased fractions of CD69hi CD103hi cells. Early after infection in the spleen, a larger fraction of Ets1-depleted P14 cells express CCR9 and α4β7. Depletion or overexpression of Ets1 in P14 cells both indicate it negatively regulates CD25 and Prdm1 expression, and promotes Tcf7 and Bcl6 expression. These results suggest that Ets1 functions early during CD8 T cell activation to establish the normal pattern of TCIRC and TRM cell differentiation during acute viral infection.

Inhibition of Arid1a increases stem/progenitor cell-like properties of liver cancer

Tumor progression in colorectal cancer (CRC) is driven by a subpopulation of cells with tumor-initiating cell (TIC) activity. In the past, bulk experiments provided strong evidence for functional heterogeneity of the CRC TIC compartment with distinct types of TICs driving CRC progression. However, in these retrospective experiments direct assignment of functional states to individual functionally relevant cell types in CRC has not been possible. We here asked whether functional states of individual cells can be assigned to specific CRC cell subpopulations. Therefore, 4,663 single-cell profiles were generated by single-cell RNA-sequencing (scRNA-seq) of patient-derived CRC spheroid cultures (n=12). Non-negative matrix factorization identified 13 gene expression modules linked to cell states or cell types that resemble differentiation states of normal intestinal epithelial cells. To evaluate their presence in CRC patient tumors, the identified transcriptional programs were applied to publicly available expression data (TCGA cohort; n=459). Clustering of samples based on the contribution of individual expression signatures revealed six patient clusters with significantly different progression-free survival (p=0.043) and a trend towards different overall survival (p=0.12), indicating a relevance for patient outcome. Comparing cell state meta-signature expression in cell type-associated subpopulations revealed differences in functional states, i.e. proliferative and metabolic activity. Interestingly, stem-like cells showed significantly increased oxidative phosphorylation (OXPHOS) and decreased glycolysis scores, suggesting a link between metabolic states and cell differentiation. scRNA-seq of patient-derived organoid and xenograft models as well as primary tumors revealed similar trends. To assess whether metabolic states are linked to TIC activity, CRC spheroid cells were sorted according to mitochondrial membrane potential (MMP), and sphere forming cell (SFC) as well as TIC frequency were assessed by limiting dilutions. Strikingly, MMPhigh cells exhibited a pronounced increase of SFC frequencies in vitro compared to MMPlow cells in 80% of patients (n=5). In addition, TIC frequencies were strongly increased in the MMPhigh compared to the MMPlow subpopulation in vivo (n=2; 1/46,535 vs. 1/211,305 and 1/249 vs. 1/2,089), demonstrating that TIC activity relies on OXPHOS. In line with this, pretreatment with the OXPHOS inhibitor CCCP decreased SFC frequencies (n=3), indicating targetable metabolic vulnerability of CRC spheroid cells. In summary, we here show that transcriptional heterogeneity identifies functional states during CRC TIC differentiation. Targeting context-dependent regulation of tumor cell differentiation might unravel novel vulnerabilities for therapeutic intervention in human CRC. Citation Format: Martina K. Zowada, Stephan M. Tirier, Sebastian M. Dieter, Teresa Krieger, Ava Oberlack, Robert L. Chua, Mario Huerta, Foo Wei Ten, Karin Laaber, Jeongbin Park, Katharina Jechow, Torsten Müller, Mathias Kalxdorf, Mark Kriegsmann, Katharina Kriegsmann, Friederike Herbst, Jeroen Krijgsveld, Martin Schneider, Roland Eils, Hanno Glimm, Christian Conrad, Claudia R. Ball. Transcriptional heterogeneity identifies functional states of tumor-initiating cell differentiation in human colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1491.

Tissue localization is a critical determinant of T cell immunity. CD8+ T cells are contact-dependent killers, which requires them to physically be within the tissue of interest to kill peptide-MHC class I-bearing target cells. Following their migration and extravasation into tissues, T cells receive many extrinsic cues from the local microenvironment, and these signals shape T cell differentiation, fate and function. Because major organ systems are variable in their functions and compositions, they apply disparate pressures on T cells to adapt to the local microenvironment. Additional complexity arises in the context of malignant lesions (either primary or metastatic), and this has made understanding the factors that dictate T cell function and longevity in tumours challenging. Moreover, T cell differentiation state influences how cues from the microenvironment are interpreted by tissue-infiltrating T cells, highlighting the importance of T cell state in the context of tissue biology. Here, we review the intertwined nature of T cell differentiation state, location, survival and function, and explain how dysfunctional T cell populations can adopt features of tissue-resident memory T cells to persist in tumours. Finally, we discuss how these factors have shaped responses to cancer immunotherapy.

SPH7854, a gut-limited RORγt antagonist, ameliorates TNBS-induced experimental colitis in rat

Defining the molecular dynamics associated with T cell differentiation enhances our understanding of T cell biology and opens up new possibilities for clinical implications. In this study, we investigated the dynamics of CD5 expression in CD8+ T cell differentiation and explored its potential clinical uses. Using PBMCs from 29 healthy donors, we observed a stepwise decrease in CD5 expression as CD8+ T cells progressed through the differentiation stages. Interestingly, we found that CD5 expression was initially upregulated in response to T cell receptor stimulation, but diminished as the cells underwent proliferation, potentially explaining the differentiation-associated CD5 downregulation. Based on the proliferation-dependent downregulation of CD5, we hypothesized that relative CD5 expression could serve as a marker to distinguish the heterogeneous CD8+ T cell population based on their proliferation history. In support of this, we demonstrated that effector memory CD8+ T cells with higher CD5 expression exhibited phenotypic and functional characteristics resembling less differentiated cells compared to those with lower CD5 expression. Furthermore, in the retrospective analysis of PBMCs from 30 non-small cell lung cancer patients, we found that patients with higher CD5 expression in effector memory T cells displayed CD8+ T cells with a phenotype closer to the less differentiated cells, leading to favorable clinical outcomes in response to immune checkpoint inhibitor (ICI) therapy. These findings highlight the dynamics of CD5 expression as an indicator of CD8+ T cell differentiation status, and have implications for the development of predictive biomarker for ICI therapy.

Decision letter: Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study

LRF Regulates Self-Renewal of Hematopoietic Stem Cells by Blocking Notch1-Mediated T Cell Differentiation

BackgroundPeripheral induced regulatory T cells (iTreg) play an important role for immune homeostasis. Dysregulation of iTreg differentiation is frequently associated with autoimmune diseases or tumors. Tumor necrosis factor (TNF) α...

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.