Memory CD8+ T cells induce precocious effector differentiation of naive T cells through quorum sensing-like behavior
ABSTRACT: Some unicellular organisms exhibit collective decision-making through intercellular communication once a quorum of members sense an environmental stress. Whether T cells at different states of differentiation may also synchronize their behavior on a population basis through direct interactions remains unclear. We report that memory CD8+ T cells (TMem) directly interact with naive T cells (TN) during priming, affecting the phenotypic, functional, transcriptional and metabolic differentiation of TN-derived progeny. This previously unrecognized, contact and concentration-dependent interaction between naive (TN) and memory CD8+ T cells (TMem) directly enhanced TN effector differentiation through non-apoptotic Fas signaling resulting in downstream Akt pathway activation. TN primed with TMem exhibited significantly impaired persistence and antitumor activity compared with TN primed alone. Disruption of FasL-Fas signaling in TN cells limited differentiation and enhanced anti-tumor immunity while provision of exogenous FasL in the absence of TMem impaired anti-tumor immunity by augmenting TN differentiation. These findings reveal that the full therapeutic potential of TN-derived cells for adoptive immunotherapy requires physical separation from TMem prior to priming or antagonism of Fas-signaling. FACS-sorted in vitro differentiated TMem samples were without stimulation (0 hours) (n=3), and FACS-sorted in vitro differentiated TMem samples were stimulated using CD3-specific and CD28-specific antibodies and IL-2 for 18 (n=3) and 96 (n=3) hours. FACS-sorted naive Pmel-1 CD8+ T cell samples were without stimulation (0 hours) (n=3), and FACS-sorted TN-derived Pmel-1 CD8+ T cell samples were stimulated using CD3-specific and CD28-specific antibodies and IL-2 for 18 (n=3) and 96 (n=3) hours. FACS-sorted TN-derived Pmel-1 CD8+ T cell samples were stimulated in the presence of TMem using CD3-specific and CD28-specific antibodies and IL-2 for 18 (n=3) and 96 (n=3) hours. FACS-sorted in vitro differentiated TMem samples were stimulated in the presence of TN using CD3-specific and CD28-specific antibodies and IL-2 for 18 (n=3) and 96 (n=3) hours. All samples were done in triplicate as biological repeats.
Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation Magnetically sorted untouched CD8+CD45R0-T cells from three different donors (named A, B and C) unstimulated or stimulated with IFN alpha 2b or with anti-CD3/CD28 beads alone or along with IFN alpha 2b or IFN alpha 5 for 48 hours.
Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation Overall design: Magnetically sorted untouched CD8+CD45R0-T cells from three different donors (named A, B and C) unstimulated or stimulated with IFN alpha 2b or with anti-CD3/CD28 beads alone or along with IFN alpha 2b or IFN alpha 5 for 48 hours.
Project description:Memory T cells (TM) play a prominent role in protective and auto-immunity because they mount a more effective response than naïve T cells (TN), by rapidly expressing a large number of immune response genes upon stimulation. Previous studies have shown a correlation between increased histone acetylation levels and rapid gene expression at specific loci in CD8+ TM. However, the underlying mechanisms of histone acetylation remodeling in TM and its role in regulating global gene expression are not well defined, particularly in human TM cells. In this study, we examined the global level of histone acetylation and the expression of histone acetyltransferases and deacetylases , and found no differences between human CD8+ TN and TM cells. Through Genomegenome-wide mapping of the histone acetyltransferase p300 in human CD8+ TN and TM cells, recruitment of p300 was found to be positively correlated with the expression of 213 genes in TM and 516 genes in TN cells at rest, and was positively correlated with the expression of 556 genes in TM and 330 genes in TN cells after 4 hrs’ stimulation. Importantly, genes which have positive correlations between expression levels and p300 occupancy in TM were significantly enriched in effector functions of CD8+ T cells, while those in TN cells were over-presented in nucleic acid processing and cell differentiations. At IFNG locus, specifically, the recruitment of p300 was significantly higher in CD8+ TM than TN and cells that were correlated with increased levels of histone acetylation at this locus, and robust IFN-g expression by CD8+ TM cells. Selective inhibition of p300 and deletion of p300 binding sites within the IFNG promoter resulted in decreased histone acetylation at this locus and the lower expression level of IFN-g in CD8+ TM cells. These results suggest that recruitment of the histone acetyltransferase p300 modifies histone acetylation and allows rapid expression of IFNG, and likely a large set of immune response genes, in human CD8+ TM, which in turn contribute to the enhanced functionality of human CD8+ TM cells. Overall design: Human naïve and memory T cells were isolated from peripheral blood of healthy individuals by FACS, and were triggered through CD3/CD28 signaling for 0 hr or 4 hr. Chromatin were fixed with 1% formaldehyde, fragmented by sonication, and fragments that were bound by p300 were immuno-precipitated with a p300 specific antibody. Immuno-precipitated DNA was analyzed by Illumina/Solexa ChIP-Sequencing.
Project description:We have shown that DC vaccine is superior to peptide vaccine in terms of priming and expansion of antigen-specific CD8+ T cells. DC vaccine-primed pmel-1 cells displayed better effecter functions than cells by peptide-primed cells in terms of cytokine production and externalization of cytotoxic granules. Furthermore DC vaccine-primed cells were metabolically distinct from peptide-primed cells. To confirm these findings, we performed a microarray analysis using splenic pmel-1 T cells from mice immunized with hgp100 peptide vaccine or DC vaccine. We also used splenic naïve pmel-1 T cells as a control. Overall design: C57BL/6 mice transferred with pmel-1 splenocytes were treated with hgp100 peptide vaccine or DC vaccine twice on days 0 and 14. On day 28, pmel-1 CD8+ T cells were sorted from harvested splenocytes of immunized mice. Gene expression was measured in naïve pmel-1 CD8+ T cells and sorted pmel-1 CD8+ T cells
Project description:Memory T cells (TM) play a prominent role in protection and auto-immunity due to their ability to mount a more effective response than naïve T cells (TN). However, the molecular mechanisms underlying enhanced functionality of TM are not well defined, particularly in human TM. We examined the global gene expression profiles of human CD8+ TN and TM before and after stimulation. There were 1,284, 1,373 and 1,629 differentially expressed genes between TN and TM at 0 hr, 4 hr and 24 hr after stimulation, respectively, with more genes expressed to higher levels in TM. Genes rapidly up-regulated in TN cells were largely involved in nitrogen, nucleoside and amino acid metabolisms. In contrast, those in CD8+ TM were significantly enriched for immune-response-associated processes, including cytokine production, lymphocyte activation and chemotaxis. Multiple cytokines were rapidly up-regulated in TM cells, including effector cytokines known to be produced by CD8+ T cells and important for their functions, as well as regulatory cytokines, both pro- and anti-inflammatory, that are not typically produced by CD8+ T cells. These results provide new insights into molecular mechanisms that contribute to the enhanced functionality of human CD8+ TM and their prominent role in protection and auto-immunity. Naïve and memory phenotype CD8 T cells were purified by FACS from healthy individuals and cultured in vitro with the stimulation of anti-CD3/CD28 mAbs for 0 hr, 4 hr and 24 hr. Total RNA was purified from un-stimulated and stimulated naive and memory CD8 T cells and hybridized to individual single-color arrays.The purification and stimulation protocol was performed two independent times.
Project description:We performed an RNA-seq analysis on FACS-sorted CD4+ memory T cells (Tmem, CD4+CD25-CD45RA-) stimulated in vitro with anti-CD3/CD28 coated beads (1:5), IL-2 (100 U/mL) in the presence of a TNF-alpha inhibitor (Etanercept, ETN, 5 μg/mL) or recombinant human TNF-alpha (rhTNF-alpha, 50 ng/mL) treatment. The cells were cultured for three days at 37°C and 5% CO2. On day three Tmem were lysed (Buffer RLT supplemented with DTT, QIAGEN) and RNA extraction was performed (RNeasy Plus Micro kit), followed by sample preparation with TruSeq (polyA) mRNA kits (Illumina), RNA sequencing (carried out by an Illumina HiSeq2500), and the alignment of trimmed fastQ files to GRCh38 human reference genome. We also performed a Quality control (QC). This was done using the tool FastQC FastQC/0.11.3-Java-1.7.0_80. QC metrics were calculated for the aligned reads using Picard-tools picard/1.130-Java-1.7.0_80, CollectRnaSeqMetrics, MarkDuplicates, CollectInsertSize-Metrics and SAMtools/1.2-foss-2015b flagstat. Finally, we carried out a Differential expression gene (DEG) analysis using DESeq2, in order to evaluate the impact of ETN and rhTNF-alpha on Transcriptome profiling of stimulated CD4+ Tmem. Principal Component Analysis (PCA) was carried out to visualise the samples given their entire transcriptome and any remaining batch effects.
Project description:The activation of TLR-MyD88 (Toll like receptor- Myeloid differentiation factor 88) signaling within T cells functions as a potent costimulatory signal that boosts antitumor and antiviral responses. However, the molecular mechanisms underlying the costimulatory processes are poorly understood. We compared microarray gene analysis data between TLR1-TLR2 stimulated and unstimulated T cell receptor transgenic ‘pmel’ and MyD88-/-pmel CD8+ T cells and identified changes in the expression levels of several TNF family members. In particular, TLR-stimulation increased 4-1BB levels in pmel but not in MyD88-/-pmel T cells. A link between 4-1BB and TLR1-TLR2 signaling in CD8+ T cells was highlighted by in fact that 4-1BB-/- T cells exhibited suboptimal responses to TLR1-TLR2 agonist, but responded normally to CD28 or OX40 costimulation. Moreover, blocking 4-1BB signaling with antibodies also hindered the costimulatory effects of the TLR1-TLR2 agonist. The elevated levels of 4-1BB transcripts in TLR1-TLR2–stimulated cells were not due to increased mRNA stability nor increased histone activation but instead were associated with increased binding of p65 and c-Jun to two distinct 4-1BB promoter sites. Combining TLR1-TLR2 ligand with an agonistic anti-4-1BB antibody enhanced the antitumor activity in mice with established melanoma tumors. These studies reveal that the costimulatory effects of TLR1-TLR2 signaling in CD8+ T cells are in part mediated by 4-1BB and are important for mounting an effective antitumor immune response. CD8+ T cells from the B6.Cg-Thy1/Cy Tg(TcraTcrb)8Rest/J mice, referred to as ‘pmel’ T cells or from MyD88 knockout pmel mice (MyD88–/–pmel) were sorted. pmel and MyD88–/–pmel T cells were activated using MyD88–/– CD8 T cell-depleted splenocytes pulsed with 10ng/ml of mgp100. This was with or without 10µg/ml of Pam3CSK4. pmel or MyD88–/–pmel CD8 T cells were enriched and used for the extraction of RNA used for genomic analysis.
Project description:The epigenetic modifier TET2 plays a role in cell fate decisions in hematopeotic stem cells and CD4+ Th1 and Th17 differentiation. Here, we demonstrate that loss of TET2 promotes CD8+ memory differentiation following acute viral infection with LCMV-Armstrong. To identify early gene expression changes following TCR activation in WT versus TET2cKO CD8+ T cells, we isolated naive CD8+ T cells and activated them for three days with anti-CD3/CD28+IL-2 and performed microarray analysis. Overall design: Naive (CD44lo CD62Lhi) CD8+ T cells were isolated and sorted from WT C57BL/6 and TET2fl/flCD4Cre+ mice. These cells were activated with plate-bound anti-CD3/CD28 for 3 days in the presence of 100U/ml hIL-2. RNA from these in vitro activated CD8+ T cells were processed, amplified, labeled and hybridized to Affymetrix GeneChip MoGene 2.0 ST microarrays.
Project description:FACS-sorted CR2+ and CR2- naive and memory CD4 T cells, plus CR2+ naive CD4 T cells activated with CD3/CD28 beads, were analyzed ex-vivo on a NanoString Immune Expression Profiling panel.
Project description:Malaria is caused by infection with the Plasmodium spp. parasite, and both T and B cells are required to clear infection over more than two weeks in the absence of treatment. The mechanisms underlying development and maintenance of memory T cells (Tmem) generated by chronic infection are not well-understood, but are critical for the development of vaccines for malaria, tuberculosis and HCV. Previous work shows that fatty acid oxidation promotes memory T cell numbers and function in the late stages of resting memory T cell generation, however little is known about metabolic decision points earlier in this differentiation pathway. We have defined a pathway of differentiation for CD4 effector memory T cells defining early precursors in malaria infection. In this study, we investigate the role of metabolism in CD4 Tmem in P. chabaudi malaria infection, a model of human malaria. We observe a relative upregulation of fatty acid synthesis (FAS) genes in CD4 Tmem compared to effector T cells (Teff). Suppression of the FAS pathway, using an inhibitor of Acc1, the first enzyme in fatty acid synthesis, impairs CD4 memory T cell development in the earliest stages of differentiation. In the first three days of T cell activation inhibition of Acc1 reduces the number of Tmem precursors and concomitantly increases Teff proliferation. The early blockade of FAS also decreases parasitemia. These data suggest that the FAS pathway drives early differentiation of CD4 memory T cells, as distinct from the Teff with high rates of glycolysis, determining protection from malaria infection. Overall design: B5 TCR Tg mice were infected with P. chabaudi. Effector and memory T cells subsets were sorted at day 8 and d60 post infection respectively. Naïve T cells from uninfected mice were sorted as well. Gene expression was measured in three effector subsets, three memory subsets and and naive T cells. Two replicates were performed using different donors for each experiment.