Transcriptional Insights Into the CD8+ T Cell Response to Infection And
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RESOURCE Transcriptional insights into the CD8+ T cell response to infection and memory T cell formation J Adam Best1, David A Blair2, Jamie Knell1, Edward Yang1, Viveka Mayya2, Andrew Doedens1, Michael L Dustin2, Ananda W Goldrath1 & The Immunological Genome Project Consortium3 After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and gd T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity. The Immunological Genome (ImmGen) Project is a partnership are well established as essential regulators of gene expression by between immunologists and computational biologists with the goal CD8+ T cells during infection, including those encoded by Tbx21, of carefully and comprehensively defining gene-expression and Tcf7, Eomes, Id2, Id3 and Prdm1, yet it is likely that many additional regulatory networks in cells of the mouse immune system by highly factors that affect CD8+ T cell differentiation are yet to be described. standardized methods of sample collection and data preparation1. Such factors are more efficiently identified by unbiased methods such Here we sought to identify and track the transcriptional programs as transcriptomics. initiated in CD8+ T cells during the response to in vivo activation CD8+ T cells are known to share certain functional abilities and + Nature America, Inc. All rights reserved. Inc. Nature America, by bacterial or viral antigens. CD8 cytotoxic T cells have important transcription factors with other cells of the immune system; how- 3 roles in the clearance of intracellular pathogens and tumors. In the ever, the transcriptional relationship between CD8+ T cells and other uninfected state, a diverse repertoire of resting, naive CD8+ T cells cytolytic lymphocyte populations is not well described. The ImmGen © 201 populate peripheral lymphoid organs. After infection, CD8+ T cells Program offered a unique opportunity to address this question, given transition from quiescent, poor effector cells to metabolically active, its unmatched inventory of directly comparable transcriptomic data proliferating cells with cytolytic function and the capacity for rapid for hundreds of different types of cells of the immune system. We cytokine production. That progression is accompanied by changes in have made a systematic and temporally resolved analysis of transcrip- gene expression that reflect each stage of differentiation2–5. During tional changes that occur through the antigen-specific responses of expansion, the innate immune response induced by different patho- CD8+ T cells, from early time points of activation to the analysis of gens creates infection-specific inflammatory environments that long-term memory cells, in the context of various infection settings. influence the kinetics of T cell population expansion and the effector From these data, we have identified previously unknown clusters of differentiation and memory potential of CD8+ T cells6,7. However, coregulated genes and used network-reconstruction analyses of the the effect of such unique proinflammatory environments on tran- ImmGen Consortium to predict transcriptional activators and repres- scriptional networks and gene expression by CD8+ T cells is not sors or genes with differences in expression. These analyses allowed well understood. us to profile CD8+ T cells with differing memory potential and obtain After pathogen clearance, most CD8+ T cells die, which leaves a insights into the transcriptional processes that govern the differentia- select few with the ability to form long-term memory and to protect tion of effector and memory cell populations. the host from reinfection. Each differentiation state—naive, effec- tor, terminally differentiated effector and memory—is thought to be RESULTS orchestrated by a network of transcription factors with key down- Temporally regulated expression patterns in CD8+ T cells stream targets that enable and enforce stage-specific cellular traits. In To establish a molecular profile of pathogen-reactive CD8+ T cells confirmation of that, certain transcriptional activators or repressors over the course of infection, we transferred congenic naive OT-I T 1Division of Biological Sciences, University of California San Diego, La Jolla, California, USA. 2Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA. 3A list of members and affiliations appears at the end of the paper. Correspondence should be addressed to A.W.G. ([email protected]). Received 9 August 2012; accepted 21 December 2012; published online 10 February 2013; doi:10.1038/ni.2536 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1 RESOURCE cells (which have transgenic expression of a T cell antigen receptor the cells on days 0.5, 1 and 2 after transfer. This approach included (TCR) that recognizes a fragment of ovalbumin (OVA; amino acids a greater frequency of precursor cells (1 × 106 transferred cells) and 257–264) presented by the major histocompatibility complex mol- allowed the infection to become established so that transferred OT-I ecule H-2Kb) into C57BL/6J mice, which we then immunized with cells were rapidly recruited into the immune response. The expression OVA-expressing Listeria monocytogenes (Lm-OVA) as a model patho- of markers associated with activation and differentiation by these cells gen-associated antigen. We collected splenic CD8+ T cells on days 6, was similar to that of cells transferred at a lower precursor frequency 8, 10, 15, 45 and 100 of infection and sorted the cells to high purity for (5 × 103 transferred cells), and any differences were consistent with gene-expression profiling by the ImmGen data-generation and qual- more rapid contraction and differentiation into the memory subset ity-control pipelines (Supplementary Fig. 1a and Supplementary (Supplementary Fig. 2). We analyzed the transferred OT-I CD8+ Note 1). We transferred the minimum number of OT-I cells that still T cells by flow cytometry for expression of phenotypic markers of allowed adequate recovery of responding cells for analysis. For col- activation and/or memory. We found that expression CD127, CD62L lection on days 6 and later, we transferred 5 × 103 donor cells 1 d and CD27 was downregulated with activation, followed by reexpres- before immunization, which represented a relatively low precursor sion in memory cells, whereas the expression of CD69 and CD44 was frequency, albeit higher than the endogenous repertoire of T cells spe- uniformly upregulated, as expected (Supplementary Fig. 1b), which cific for H2-Kb–OVA peptide8,9. To gain better understanding of the indicated that all of the transferred cells were activated. changes in gene expression that occur during the earliest stages of the The number of genes with different expression in infection-exposed response after activation, before the expansion phase, we used the fol- OT-I cells versus naive OT-I cells peaked within 48 h of infection; lowing alternative approach: we first infected mice with Lm-OVA and, unexpectedly, at later time points, a greater proportion of genes with 1 d later, transferred OT-I CD8+ cells into the mice and then isolated altered expression were downregulated than were upregulated (Fig. 1a), a b –3.0 3.0 20 Expression (fold) Up ) 2 15 Down 10 × ( 10 Genes 5 0 12 24 48 6 8 10 15 45 100 Naive 12 24 48 6 8 10 15 45 100 Time (h) Time (d) Time (h) Time (d) Initial cytokine or effector Preparation for cell Cell cycle & division: Naive and late memory: Early effector, late c response: Ctla4, Ifng, division: Myc, Tnf, Id3, Myb, Hist1h3a, Cdk1, Sell, Nsg2, Slfn5, Cnr2 memory: Ly6a, Rpl, d Gzmb, Il2ra, Il2 Egr2, Cd69, Pkm2 Cdc45 Snora I II III IV V Nature America, Inc. All rights reserved. Inc. Nature America, Time (d) Time (d) Expression (fold) 3 28 577 291 84 225 6 8 6 8 6 8 6 8 6 8 N 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 100 100 100 100 100 N 6 8 10 15 45 100 N 6 8 10 15 45 100 Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Dmrta1 Unc5a © 201 Short-term effector Memory precursor: Naive or late effector or Short-term effector or Late effector or memory: Edaradd Xcl1 and memory: Id2, Bcl2, Tcf7, Il7r, Foxo3 memory: Cxcr6, Klf2, memory: Prdm1, Hif2a Tbx21, Prf1, Bhlhe40, Prss12 Yes1 Zeb2, Klrg1, S1pr5, Klf3, S1pr4, S1pr1 Cd44, Klre1, Il12rb2 Cdh1 Cxcr3, Cx3cr1, Itgam Cnrip1 Aqp9 Myo3b VI VII VIII IX X Bcl2 Dock9 –3.0 3.0 Atn1 Expression (fold) Expression (fold) 53 123 219 82 92 N 6 8 6 8 6 8 6 8 6 8 0 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 N 12 24 48 10 15 45 100 100 100 100 10 Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Time (h) Time (d) Figure 1 Gene-expression profiles associated with the activation e Glycolysis ) 100 Respiratory chain and memory formation of CD8+ T cells.