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.

Integrin Activation Controls Regulatory T Cell-Mediated Peripheral Tolerance.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and ß subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a ß integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4ß1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Talin Plays a Critical Role in the Maintenance of the Regulatory T Cell Pool.

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1fl/flCd4Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.

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.

Ubiquitylation of CD98 limits cell proliferation and clonal expansion.

CD98 heavy chain (SLC3A2) facilitates lymphocyte clonal expansion that enables adaptive immunity; however, increased expression of CD98 is also a feature of both lymphomas and leukemias and represents a potential therapeutic target in these diseases. CD98 is transcriptionally regulated and ectopic expression of the membrane-associated RING-CH (MARCH) E3 ubiquitin ligases MARCH1 or MARCH8 leads to ubiquitylation and lysosomal degradation of CD98. Here, we examined the potential role of ubiquitylation in regulating CD98 expression and cell proliferation. We report that blocking ubiquitylation by use of a catalytically inactive MARCH or by creating a ubiquitylation-resistant CD98 mutant, prevents MARCH-induced CD98 downregulation in HeLa cells. March1-null T cells display increased CD98 expression. Similarly, T cells expressing ubiquitylation-resistant CD98 manifest increased proliferation in vitro and clonal expansion in vivo. Thus, ubiquitylation and the resulting downregulation of CD98 can limit cell proliferation and clonal expansion.

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.

Symmetric Arginine Dimethylation Is Selectively Required for mRNA Splicing and the Initiation of Type I and Type III Interferon Signaling.

Alternative splicing is well understood to enhance proteome diversity as cells respond to stimuli. However, mechanistic understanding for how the spliceosome processes precursor messenger RNA (mRNA) transcripts to achieve template diversification is incomplete. We use recently developed enzymatic inhibitors of protein arginine methyltransferase 5 (PRMT5) and human naive T lymphocyte activation as a model system to uncover a precise set of mRNA transcripts that require symmetric arginine dimethylation. This methylation-dependent splicing selectivity is associated with a limited set of signaling pathways that are affected when PRMT5 is inhibited. Specifically, we identify a conserved role for symmetric arginine dimethylation in the induction of antiviral type I and type III interferon signaling following T cell receptor and pattern recognition receptor stimulation in human T lymphocytes and undifferentiated human THP-1 monocytes. Altogether, these findings reveal a mechanism by which cells may be enabled to precisely modulate transcript heterogeneity to orchestrate specific functional outcomes.

Proteasome activity regulates CD8+ T lymphocyte metabolism and fate specification.

During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.

Regulation of Asymmetric Division by Atypical Protein Kinase C Influences Early Specification of CD8(+) T Lymphocyte Fates.

Naïve CD8(+) T lymphocytes responding to microbial pathogens give rise to effector T cells that provide acute defense and memory T cells that provide long-lived immunity. Upon activation, CD8(+) T lymphocytes can undergo asymmetric division, unequally distributing factors to the nascent daughter cells that influence their eventual fate towards the effector or memory lineages. Individual loss of either atypical protein kinase C (aPKC) isoform, PKCζ or PKCλ/ι, partially impairs asymmetric divisions and increases CD8(+) T lymphocyte differentiation toward a long-lived effector fate at the expense of memory T cell formation. Here, we show that deletion of both aPKC isoforms resulted in a deficit in asymmetric divisions, increasing the proportion of daughter cells that inherit high amounts of effector fate-associated molecules, IL-2Rα, T-bet, IFNγR, and interferon regulatory factor 4 (IRF4). However, unlike CD8(+) T cells deficient in only one aPKC isoform, complete loss of aPKC unexpectedly increased CD8(+) T cell differentiation toward a short-lived, terminal effector fate, as evidenced by increased rates of apoptosis and decreased expression of Eomes and Bcl2 early during the immune response. Together, these results provide evidence for an important role for asymmetric division in CD8(+) T lymphocyte fate specification by regulating the balance between effector and memory precursors at the initiation of the adaptive immune response.

Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that ß2 and ß5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-ß signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.