Pyrimidine de novo synthesis inhibition selectively blocks effector but not memory T cell development.

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

Proliferation-competent Tcf1+ CD8 T cells in dysfunctional populations are CD4 T cell help independent.

T cell maintenance in chronic infection and cancer follows a hierarchical order. Short-lived effector CD8 T cells are constitutively replaced from a proliferation-competent Tcf1-expressing progenitor population. This occurs spontaneously at low levels and increases in magnitude upon blocking PD-1 signaling. We explore how CD4 T cell help controls transition and survival of the progenitors and their progeny by utilizing single-cell RNA sequencing. Unexpectedly, absence of CD4 help caused reductions in cell numbers only among terminally differentiated cells while proliferation-competent progenitor cells remained unaffected with regard to their numbers and their overall phenotype. In fact, upon restoration of a functional CD4 compartment, the progenitors began to regenerate the effector CD8 T cells. Thus, unlike memory T cells for which secondary expansion requires CD4 T cell help, this is not a necessity for proliferation-competent progenitor cells in dysfunctional populations. Our data therefore reveals that proliferation-competent cells in dysfunctional populations show a previously unrecognized uncoupling of CD4 T cell help that is otherwise required by conventional memory T cells.

Secondary infections rejuvenate the intestinal CD103+ tissue-resident memory T cell pool.

Resident T lymphocytes (TRM) protect tissues during pathogen reexposure. Although TRM phenotype and restricted migratory pattern are established, we have a limited understanding of their response kinetics, stability, and turnover during reinfections. Such characterizations have been restricted by the absence of in vivo fate-mapping systems. We generated two mouse models, one to stably mark CD103+ T cells (a marker of TRM cells) and the other to specifically deplete CD103- T cells. Using these models, we observed that intestinal CD103+ T cells became activated during viral or bacterial reinfection, remained organ-confined, and retained their original phenotype but failed to reexpand. Instead, the population was largely rejuvenated by CD103+ T cells formed de novo during reinfections. This pattern remained unchanged upon deletion of antigen-specific circulating T cells, indicating that the lack of expansion was not due to competition with circulating subsets. Thus, although intestinal CD103+ resident T cells survived long term without antigen, they lacked the ability of classical memory T cells to reexpand. This indicated that CD103+ T cell populations could not autonomously maintain themselves. Instead, their numbers were sustained during reinfection via de novo formation from CD103- precursors. Moreover, in contrast to CD103- cells, which require antigen plus inflammation for their activation, CD103+ TRM became fully activated follwing exposure to inflammation alone. Together, our data indicate that primary CD103+ resident memory T cells lack secondary expansion potential and require CD103- precursors for their long-term maintenance.

Origin and fine-tuning of effector CD8 T cell subpopulations in chronic infection.

Persisting stimulation can skew CD8 T cells towards a hypofunctional state commonly referred to as T cell exhaustion. This functional attenuation likely constitutes a mechanism which evolved to balance T cell mediated viral control versus overwhelming immunopathology. Here, we highlight the recent progress in defining the genetic mechanisms and factors shaping the differentiation of exhausted CD8 T cells. We review how the transcription factor Tox imposes an exhausted phenotype in the Tcf1+ progenitors and how CD4 help fine-tunes the effector subsets that emerge from this progenitor population. Both processes critically shape the spectrum of effector function performed by CD8 T cells and the level of resulting virus control. Finally, we discuss how these insights can be exploited to boost the immune response in chronic infection and cancer.

Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells.

Single-cell RNA sequencing in principle offers unique opportunities to improve the efficacy of contemporary T-cell based immunotherapy against cancer. The use of high-quality single-cell data will aid our incomplete understanding of molecular programs determining the differentiation and functional heterogeneity of cytotoxic T lymphocytes (CTLs), allowing for optimal therapeutic design. So far, a major obstacle to high depth single-cell analysis of CTLs is the minute amount of RNA available, leading to low capturing efficacy. Here, to overcome this, we tailor a droplet-based approach for high-throughput analysis (tDrop-seq) and a plate-based method for high-performance in-depth CTL analysis (tSCRB-seq). The latter gives, on average, a 15-fold higher number of captured transcripts per gene compared to droplet-based technologies. The improved dynamic range of gene detection gives tSCRB-seq an edge in resolution sensitive downstream applications such as graded high confidence gene expression measurements and cluster characterization. We demonstrate the power of tSCRB-seq by revealing the subpopulation-specific expression of co-inhibitory and co-stimulatory receptor targets of key importance for immunotherapy.

Enabling single-cell trajectory network enrichment.

Single-cell sequencing (scRNA-seq) technologies allow the investigation of cellular differentiation processes with unprecedented resolution. Although powerful software packages for scRNA-seq data analysis exist, systems biology-based tools for trajectory analysis are rare and typically difficult to handle. This hampers biological exploration and prevents researchers from gaining deeper insights into the molecular control of developmental processes. Here, to address this, we have developed Scellnetor; a network-constraint time-series clustering algorithm. It allows extraction of temporal differential gene expression network patterns (modules) that explain the difference in regulation of two developmental trajectories. Using well-characterized experimental model systems, we demonstrate the capacity of Scellnetor as a hypothesis generator to identify putative mechanisms driving haematopoiesis or mechanistically interpretable subnetworks driving dysfunctional CD8 T-cell development in chronic infections. Altogether, Scellnetor allows for single-cell trajectory network enrichment, which effectively lifts scRNA-seq data analysis to a systems biology level.

Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer.

PURPOSE: Radioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines. METHODS: The human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H2DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion. RESULTS: The acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines. CONCLUSIONS: Our data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype.

Molecular RNA Correlates of the SOFA Score in Patients with Sepsis.

The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system); MMP9 and PDE4B (nervous system); PPARG (cardiovascular system); AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury); GFER/ALR (liver); and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity.