Modulation of Let-7 Mirnas Controls the Differentiation of Effector CD8 T Cells
Total Page：16
File Type：pdf, Size：1020Kb
1 Modulation of let-7 miRNAs controls the differentiation of effector CD8 T cells 2 3 4 Alexandria C. Wells1, Keith A. Daniels2, Constance C. Angelou1, Eric Fagerberg1, Amy 5 S. Burnside1, Michele Markstein3, Dominique Alfandari1, Raymond M. Welsh2, Elena L. 6 Pobezinskaya1*, Leonid A. Pobezinsky1* 7 8 1Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, 9 MA 01003 10 2Department of Pathology, University of Massachusetts Medical School, MA 01605 11 3Department of Biology, University of Massachusetts, Amherst, MA 01003. 12 13 14 Running title: let-7 regulates CTL-mediated immune responses 15 16 Keywords: CTL; naive; Myc; Eomes; TCR; Metabolism 17 18 *Correspondence to: 19 Leonid A. Pobezinsky, Department of Veterinary and Animal Sciences, University 20 of Massachusetts, Amherst, MA 01003 21 Tel: 413-545-2393 22 Fax: 413-545-6326 23 Email: [email protected] 24 Elena L. Pobezinskaya, Department of Veterinary and Animal Sciences, 25 University of Massachusetts, Amherst, MA 01003 26 Tel: 413-545-2405 27 Email: [email protected] 28 29 Abstract 30 The differentiation of naïve CD8 T cells into effector cytotoxic T lymphocytes upon 31 antigen stimulation is necessary for successful anti-viral, and anti-tumor immune 32 responses. Here, using a mouse model, we describe a dual role for the let-7 microRNAs 33 in the regulation of CD8 T cell responses, where maintenance of the naïve phenotype in 34 CD8 T cells requires high levels of let-7 expression, while generation of cytotoxic T 35 lymphocytes depends upon T cell receptor mediated let-7 downregulation. Decrease of 36 let-7 expression in activated T cells enhances clonal expansion and the acquisition of 37 effector function through derepression of the let-7 targets, including Myc and 38 Eomesodermin. Ultimately, we have identified a novel let-7 mediated mechanism, which 39 acts as a molecular brake controlling the magnitude of CD8 T cell responses. 40 41 42 Introduction 43 44 CD8+ T cells are responsible for the rapid clearance of virally infected, and 45 cancerous cells in the organism. Prior to encounter with antigen, naïve CD8 T cells 46 exhibit a quiescent state that is characterized by very low rates of proliferation, and a 47 “quiet” transcriptional landscape with no expression of any effector molecules. After 48 antigen recognition, activated CD8 T cells undergo blastogenesis and rapid clonal 49 expansion, which is followed by differentiation into effector cytotoxic T lymphocytes 50 (CTLs), and memory T cells that are both capable of producing effector cytokines and 51 killing target cells. These changes in T cells are accompanied by metabolic 52 reprograming from oxidative phosphorylation to aerobic glycolysis that provides energy 53 and larger amounts of the biomacromolecular intermediates needed to support growth 1. 54 The productive differentiation of CD8 T cells requires three definitive signals; one 55 is from the T cell receptor (TCR) during antigen recognition, the second is from co- 56 stimulation, and the third is due to cytokines. It has been extensively studied how these 57 signals execute the CD8 T cell differentiation program by activation of a complex 58 network of transcription factors such as Myc, Notch-1, Tbet, Eomes, Blimp-1, Zbtb32, 59 Runx3, and Zeb2 2-5. However, the global regulation of these processes is not yet fully 60 understood. 61 Post-transcriptional regulatory mechanisms provide broad regulation of gene 62 expression. The most efficient post-transcriptional regulatory machinery involves RNA 63 interference mediated by microRNAs (miRNAs), small non-coding RNAs that target 64 mRNA in a sequence-specific manner to prevent protein synthesis, and the expression 65 of which is both inducible, and tissue- specific 6,7. The importance of this type of 1 66 regulation for T cells was first demonstrated through the ectopic expression of various 67 miRNAs during hematopoiesis 8. Later, it was found that miRNA depletion achieved by 68 a T cell- specific deletion of Dicer, a critical enzyme involved in the biogenesis of mature 69 miRNAs, resulted in severe defects, including aberrant proliferation, differentiation, and 70 function of T lymphocytes 9-11. Further research corroborated these earlier studies, 71 identifying the role of specific miRNAs in these processes. MiR-181 was shown to 72 modulate TCR sensitivity and signal strength in developing and mature T cells 12,13. In 73 CD4 T cells, miR-29 was shown to control the T helper response through the direct 74 targeting of IFN-γ mRNA 14, as well as the transcription factors T-bet and Eomes 15. In 75 CD8 T cells, the upregulation of miR-130/301 16 and the miR-17-92 cluster 15,17,18 was 76 demonstrated to be essential for the initiation of differentiation upon antigen stimulation. 77 Let-7 miRNAs are the largest and most abundant family of miRNAs in 78 lymphocytes, yet very little is know about their function. It has been shown that let-7 79 miRNAs are needed for the differentiation and function of natural killer T cells, an 80 innate-like subset of T cells 19,20. It has also been suggested that let-7 miRNAs may 81 regulate T helper responses 21,22, and play a role in the suppressive function of Tregs 23. 82 However, the extent to which let-7 miRNAs regulate the differentiation or function of 83 CD8 T cells has yet to be explored. 84 In the present study, we examine the role of let-7 miRNA expression in naïve and 85 differentiating CD8 T cells. We found that in CD8 T cells high levels of let-7 miRNAs are 86 necessary to maintain the naïve phenotype, while TCR-mediated down-regulation of let- 87 7 levels in activated cells is critical for the effective differentiation and function of CTLs. 88 In fact, experimentally forced let-7 expression severely impairs the proliferation and 2 89 differentiation of CD8 T cells, while let-7 deficiency significantly enhances the cytotoxic 90 function of CTLs, and consequently immune responses in vivo. Given these findings, we 91 propose a model in which let-7 acts as a molecular hub by converting the strength of 92 TCR signaling into the strength of CD8 T cell function. 93 94 Results 95 96 let-7 miRNA expression maintains the quiescent state in naïve CD8 T cells 97 98 To explore the potential regulatory role of let-7 miRNAs in CD8 T cells, the 99 expression levels of let-7 miRNA family members in naïve and activated CD8 T cells 100 were determined and normalized to two different house keeping RNAs. Surprisingly, the 101 initially very high expression of let-7 miRNAs in naïve CD8 T cells was reduced by TCR 102 signaling, and this downregulation was proportional to the strength and duration of TCR- 103 stimulation, regardless of house keeping RNA used (Figure 1A, B and Figure 1—figure 104 supplement 1 and Figure 1—figure supplement 2A). As a specificity control, we 105 confirmed the upregulation of miR-17 from the miR-17-92 locus (Figure 1—figure 106 supplement 2B) that is induced upon T cell activation17,18. Taken together, these results 107 suggest that TCR-mediated signaling inhibits the expression of let-7 miRNAs during T 108 cell activation. 109 To determine the functional significance of let-7 expression in naïve CD8 110 lymphocytes, we examined CD8 T cells from P14+Lin28TgRag2-/- mice where T cell- 111 specific expression of the Lin28 protein blocks let-7 biogenesis19,24, inhibiting let-7 112 expression, and P14 is a monoclonal T cell receptor specific to the lymphocytic 113 choriomeningitis virus (LCMV) peptide gp33-41, presented in the context of H-2Db 3 114 molecules (Figure 1—figure supplement 3A). In comparison to the P14+ wild type 115 counterparts, P14+Lin28Tg CD8 T cells were significantly larger in size, with a 116 dramatically increased proportion of Ki67 positive cells 25 (Figure 1C, D). Consistent 117 with the increased proportion of Ki67 positive cells, P14+Lin28Tg mice had a 118 significantly higher frequency of BrdU positive cells in both the spleen and lymph nodes, 119 as compared to wild type P14+ mice (Figure 1E). In addition, surface expression of T 120 cell activation markers, such as the IL-2 receptor beta-chain (CD122), and CD44 was 121 also increased, while cells remained CD25 negative (Figure 1—figure supplement 3B). 122 Thus, these results suggest that the expression of let-7 miRNAs may maintain the 123 quiescent state in naïve CD8 T cells. 124 125 let-7 miRNA expression in CTLs affects both the anti-viral and anti-tumor immune 126 responses 127 TCR stimulation of naive T cells leads to a rapid loss of the quiescent state and 128 differentiation into effector cells. Given that the expression of let-7 miRNAs, which is 129 critical for the maintenance of naïve CD8 T cells, is inhibited by TCR signaling (Figure 130 1A, B), we hypothesized that the downregulation of let-7 miRNAs in response to TCR 131 stimulation is necessary for the differentiation and function of effector CD8 T cells 132 (Figure 2A). 133 To determine whether TCR-mediated downregulation of let-7 miRNAs is required 134 for CD8 T cell differentiation in vivo, we analyzed the fate of P14+ CD8 T cells with 135 forced let-7 expression in response to acute viral infection with LCMV Armstrong. The 136 doxycycline- inducible let-7g transgene 26 maintains let-7g miRNA expression in 4 137 lymphocytes in the presence of doxycycline, even after TCR stimulation (Figure 2— 138 Figure supplement 1A). Donor CD45.2+CD8+ T cells from P14+ and P14+ let-7 139 transgenic (let-7Tg) mice were adoptively transferred into host congenic wild type 140 CD45.1+ mice that were concurrently infected with LCMV, and the differentiation state of 141 P14+ cells was assessed seven days post- injection.