Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing.

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8+ T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

Early specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single-cell gene-expression analyses.

T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combined single-cell gene-expression analyses with 'machine-learning' approaches to trace the transcriptional 'roadmap' of individual CD8(+) T lymphocytes throughout the course of an immune response in vivo. Gene-expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may have been influenced by asymmetric partitioning of the receptor for interleukin 2 (IL-2Rα) during mitosis. Our findings emphasize the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection.

Asymmetric Cell Division in T Lymphocyte Fate Diversification.

Immunological protection against microbial pathogens is dependent on robust generation of functionally diverse T lymphocyte subsets. Upon microbial infection, naïve CD4(+) or CD8(+) T lymphocytes can give rise to effector- and memory-fated progeny that together mediate a potent immune response. Recent advances in single-cell immunological and genomic profiling technologies have helped elucidate early and late diversification mechanisms that enable the generation of heterogeneity from single T lymphocytes. We discuss these findings here and argue that one such mechanism, asymmetric cell division, creates an early divergence in T lymphocyte fates by giving rise to daughter cells with a propensity towards the terminally differentiated effector or self-renewing memory lineages, with cell-intrinsic and -extrinsic cues from the microenvironment driving the final maturation steps.

Regulation of asymmetric division and CD8+ T lymphocyte fate specification by protein kinase Cζ and protein kinase Cλ/ι.

During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.

Sex differences in CD8+ T cell responses during adaptive immunity.

Biological sex is an important variable that influences the immune system's susceptibility to infectious and non-infectious diseases and their outcomes. Sex dimorphic features in innate and adaptive immune cells and their activities may help to explain sex differences in immune responses. T lymphocytes in the adaptive immune system are essential to providing protection against infectious and chronic inflammatory diseases. In this review, T cell responses are discussed with focus on the current knowledge of biological sex differences in CD8+ T cell mediated adaptive immune responses in infectious and chronic inflammatory diseases. Future directions aimed at investigating the molecular and cellular mechanisms underlying sex differences in diverse T cell responses will continue to underscore the significance of understanding sex differences in protective immunity at the cellular level, to induce appropriate T cell-based immune responses in infection, autoimmunity, and cancer. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology Infectious Diseases > Molecular and Cellular Physiology.

Comparative analysis of poxvirus orthologues of the vaccinia virus E3 protein: modulation of protein kinase R activity, cytokine responses, and virus pathogenicity.

Poxviruses are important human and animal pathogens that have evolved elaborate strategies for antagonizing host innate and adaptive immunity. The E3 protein of vaccinia virus, the prototypic member of the orthopoxviruses, functions as an inhibitor of innate immune signaling and is essential for vaccinia virus replication in vivo and in many human cell culture systems. However, the function of orthologues of E3 expressed by poxviruses of other genera with different host specificity remains largely unknown. In the present study, we characterized the E3 orthologues from sheeppox virus, yaba monkey tumor virus, swinepox virus, and myxoma virus for their ability to modulate protein kinase R (PKR) function, cytokine responses and virus pathogenicity. We found that the E3 orthologues of myxoma virus and swinepox virus could suppress PKR activation and interferon (IFN)-induced antiviral activities and restore the host range function of E3 in HeLa cells. In contrast, the E3 orthologues from sheeppox virus and yaba monkey tumor virus were unable to inhibit PKR activation. While the sheeppox orthologue was unable to restore the host range function of E3, the yaba monkey tumor virus orthologue partially restored E3-deficient vaccinia virus replication in HeLa cells, correlated with its ability to suppress IFN-induced antiviral activities. Moreover, poxvirus E3 orthologues show varying ability to inhibit the induction of antiviral and proinflammatory cytokines. Despite these in vitro results, none of the E3 orthologues tested was capable of restoring pathogenicity to E3-deficient vaccinia virus in vivo.

Vaccinia virus E3 suppresses expression of diverse cytokines through inhibition of the PKR, NF-kappaB, and IRF3 pathways.

The vaccinia virus double-stranded RNA binding protein E3 has been demonstrated to inhibit the expression of cytokines, including beta interferon (IFN-beta) and tumor necrosis factor alpha (TNF-alpha). However, few details regarding the molecular mechanisms of this inhibition have been described. Using real-time PCR arrays, we found that E3 suppressed the induction of a diverse array of cytokines representing members of the IFN, interleukin (IL), TNF, and transforming growth factor cytokine families. We discovered that the factor(s) responsible for the induction of IL-6, TNF-alpha, and inhibin beta A (INHBA) was associated with the early and late phases of virus infection. In contrast, the factor(s) which regulates IFN-beta induction was associated with the late phase of replication. We have found that expression of these cytokines can be induced by transfection of cells with RNA isolated from vaccinia virus-infected cells. Moreover, we provide evidence that E3 antagonizes both PKR-dependent and PKR-independent pathways to regulate cytokine expression. PKR-dependent activation of p38 and NF-kappaB was required for vaccinia virus-induced INHBA expression, whereas induction of TNF-alpha required only PKR-dependent NF-kappaB activation. In contrast, induction of IL-6 and IFN-beta was largely PKR independent. IL-6 induction is regulated by NF-kappaB, while IFN-beta induction is mediated by IFN-beta promoter stimulator 1 and IFN regulatory factor 3/NF-kappaB. Collectively, these results indicate that E3 suppresses distinct but interlinked host signaling pathways to inhibit the expression of a diverse array of cytokines.

Vaccinia virus K1L and C7L inhibit antiviral activities induced by type I interferons.

Cellular tropism of vaccinia virus (VACV) is regulated by host range genes, including K1L, C7L, and E3L. While E3L is known to support viral replication by antagonizing interferon (IFN) effectors, including PKR, the exact functions of K1L and C7L are unclear. Here, we show that K1L and C7L can also inhibit antiviral effectors induced by type I IFN. In human Huh7 and MCF-7 cells, a VACV mutant lacking both K1L and C7L (vK1L-C7L-) replicated as efficiently as wild-type (WT) VACV, even in the presence of IFN. However, pretreating the cells with type I IFN, while having very little effect on WT VACV, blocked the replication of vK1L-C7L- at the step of intermediate viral gene translation. Restoring either K1L or C7L to vK1L(-)C7L(-) fully restored the IFN resistance phenotype. The deletion of K1L and C7L from VACV did not affect the ability of the virus to inhibit IFN signaling or its ability to inhibit the phosphorylation of PKR and the alpha subunit of eukaryotic initiation factor 2, indicating that K1L and C7L function by antagonizing an IFN effector(s) but with a mechanism that is different from those of IFN antagonists previously identified for VACV. Mutations of K1L that inactivate the host range function also rendered K1L unable to antagonize IFN, suggesting that K1L supports VACV replication in mammalian cells by antagonizing the same antiviral factor(s) that is induced by IFN in Huh7 cells.

Single-cell analysis of CD8 T lymphocyte diversity during adaptive immunity.

An effective adaptive immune response to microbial infection relies on the generation of heterogeneous T lymphocyte fates and functions. CD8 T lymphocytes play a pivotal role in mediating immediate and long-term protective immune responses to intracellular pathogen infection. Systems-based analysis of the immune response to infection has begun to identify cell fate determinants and the molecular mechanisms underpinning CD8 T lymphocyte diversity at single-cell resolution. Resolving CD8 T lymphocyte heterogeneity during adaptive immunity highlights the advantages of single-cell technologies and computational approaches to better understand the ontogeny of CD8 T cellular diversity following infection. Future directions of integrating single-cell multiplex approaches capitalize on the importance of systems biology in the understanding of immune CD8 T cell differentiation and functional diversity. This article is categorized under: Physiology > Mammalian Physiology in Health and Disease Biological Mechanisms > Cell Fates.

Single-Cell Transcriptomics of Immune Cells: Cell Isolation and cDNA Library Generation for scRNA-Seq.

Single-cell RNA-sequencing (scRNA-seq) enables a comprehensive analysis of the transcriptome of individual cells by next-generation sequencing. ScRNA-seq offers an unbiased approach to investigate the cellular heterogeneity and dynamics of diverse biological systems, including the immune system. Optimization of the technical procedures performed prior to RNA-seq analysis is imperative to the success of a scRNA-seq experiment. Here, three major experimental procedures are described: (1) the isolation of immune CD8a+ T cells from primary murine tissue, (2) the generation of single-cell cDNA libraries using the 10× Genomics Chromium Controller and the Chromium Single Cell 3' Solution, and (3) cDNA library quality control. In this protocol, CD8a+ T cells are isolated from murine spleen tissue, but any cell type of interest can be enriched and used for single-cell cDNA library generation and subsequent RNA-seq experiments.

An OTX2-PAX3 signaling axis regulates Group 3 medulloblastoma cell fate.

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.

RNA species generated in vaccinia virus infected cells activate cell type-specific MDA5 or RIG-I dependent interferon gene transcription and PKR dependent apoptosis.

RNA species produced during virus replication are pathogen-associated molecular patterns (PAMPs) triggering cellular innate immune responses including induction of type I interferon expression and apoptosis. Pattern recognition receptors (PRRs) for these RNAs include the retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation associated gene 5 (MDA5) and the dsRNA dependent protein kinase (PKR). Currently, poxvirus PAMPs and their associated PRRs are not well characterized. We report that RNA species generated in vaccinia infected cells can activate MDA5 or RIG-I dependent interferon-ß (IFN-ß) gene transcription in a cell type-specific manner. These RNA species also induce the activation of apoptosis in a PKR dependent, but MDA5 and RIG-I independent, manner. Collectively our results demonstrate that RNA species generated during vaccinia virus replication are major PAMPs activating apoptosis and IFN-ß gene transcription. Moreover, our results delineate the signaling pathways involved in the recognition of RNA-based poxvirus PAMPs.

Antagonizing activity of vaccinia virus E3L against human interferons in Huh7 cells.

The E3L protein of vaccinia virus (VV) is well known for its capacity to evade cellular innate antiviral immunity related to interferon (IFN), for example PKR and RNaseL mediated antiviral activities. However, due to the limited range of cells that support VV E3L deletion mutant replication, the full capacity of E3L inhibiting the innate immune response induced by IFNs remains to be examined. In this report, the inhibition activity of VV E3L against a wide spectrum of human IFNs, including type I IFNs (12 IFN-alpha subtypes, IFN-beta, and IFN-omega), and type II IFN (gamma), was comparatively examined using the Copenhagen strain E3L deletion mutant and its revertant control virus in a human hepatoma cell line, Huh7. Deletion of the E3L open reading frame rendered the mutant VV sensitive to all types of IFNs, while the revertant VV was strongly resistant to these treatments. Furthermore, we show that the inhibition of VV E3L deletion mutant by IFN occurs at the stage of intermediate gene translation, while the expression of early genes and transcription of intermediate genes are largely unaffected. Using specific siRNAs to suppress the classical IFN-induced antiviral pathways, we found that PKR is the key factor modulated by E3L, while the RNaseL and MxA pathways play limited roles in this Huh7 cell system. Thus, our data demonstrates that VV E3L can mediate strong inhibition activity against all human type I and type II IFNs, mainly through modulation of the PKR pathway in Huh7 cells.