Unknown,Transcriptomics,Genomics,Proteomics

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Immediate dysfunction of vaccine-elicited CD8+ T cells primed in the absence of CD4+ T cells

ABSTRACT: CD4+ T cell help is critical for optimal CD8+ T cell expansion after priming in many experimental systems. However, a role for CD4+ T cells in regulating the initial steps of CD8+ T cell effector differentiation is not well established. Here we demonstrate that absence of CD4+ T cells at the time of replication-incompetent adenovirus vector immunization of C57BL/6 mice led to immediate CD8+ T cell dysfunction characteristic of exhaustion at the first detectable timepoints as well as impaired expansion of antigen-specific CD8+ T cells. The absence of CD4+ T cell help resulted in antigen-specific CD8+ T cells that had reduced ex vivo cytotoxicity and decreased capacity to produce IFN-Î³ and TNF-Î±. CD8+ T cells primed in the absence of CD4+ T cells expressed elevated levels of the inhibitory receptors PD-1, LAG-3, and Tim-3, and these cells exhibited transcriptomic exhaustion profiles by gene set enrichment analysis. This dysfunctional state was imprinted within 3 days of immunization and could not be reversed by provision of CD4+ T cell help after priming. Partial rescue of unhelped CD8+ T cell expansion and effector differentiation could be achieved by PD-1 pathway blockade or recombinant IL-2 administration. This study identifies a novel, previously undescribed role of CD4+ T cells to prevent immediate dysfunction and features of exhaustion in CD8+ T cells following antigen priming. Splenic AL11-specific CD8 T cells from mice immunized with Ad5HVR48(1-7)-SIV Gag and treated with anti-CD4 antibody or not were purified by FACS on day 14 post-immunization

ORGANISM(S): Mus musculus

SUBMITTER: Nicholas Provine

PROVIDER: E-GEOD-73001 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CD4+ ‘helper’ T cells (TH) are pivotal for the generation and maintenance of CD8+ T cell responses. ‘Helped’ CD8+ T cells receive signals during priming that prevent the induction of the pro-apoptotic molecule TRAIL during reactivation, thereby enabling robust secondary expansion. Conversely, ‘helpless’ CD8+ T cells primed in the absence of TH induce TRAIL expression upon restimulation and undergo activation-induced cell death (AICD). We have investigated the molecular basis for the differential regulation of TRAIL in helped versus helpless CD8+ T cells by comparing their transcriptional profiles, and have identified a transcriptional corepressor, NGFI-A binding protein 2 (Nab2) that is selectively induced in helped CD8+ T cells. Enforced expression of Nab2 prevents TRAIL induction upon restimulation of primary helpless CD8+ T cells, and expression of a dominant-negative form of Nab2 in helped CD8+ T cells impairs their secondary proliferative response that is reversible by TRAIL blockade. Finally, we observe that the CD8+ T cell autocrine growth factor interleukin-2 (IL-2) coordinately increases Nab2 expression and decreases TRAIL expression. These findings identify Nab2 as a mediator of TH-dependent CD8+ T cell memory responses through regulation of TRAIL and promotion of secondary expansion, and suggest a mechanism through which this operates. Helped and helpless E1B192-200-specific CD8+ T cells were generated as previously described16. Briefly, C57BL/6J mice were treated with 100 ug GK1.5 intraperitoneally (helpless) or left untreated (helped) prior to subcutaneous immunization with 1x107 irradiated (3000 rad) TAP-/-Ad5E1-MEC. 3 days post immunization, all mice were treated with 100 ug GK1.5. Spleens and draining lymph nodes were harvested 7 days post immunization, and CD8+ T cells were purified with the CD8 negative isolation kit (Miltenyi) according to the manufacturer’s protocol. Purity was 88-95%. Microarray and data analysis Purified CD8+ T cells from helped and helpless mice were sorted on CD44hi expression by flow cytometry. Cells were restimulated for 4h with 2ug/ml E1B192-200 peptide in the presence of 20uM qVD-OPh (R&D Systems). RNA was extracted using TriZol (Gibco BRL) according to the manufacturer's instructions. 5ug of total RNA was used to generate cDNA with RT Superscript III (Invitrogen). cDNA was indirectly labeled using Cyanine 3 and Cyanine 5 (Amersham). Labeled samples were purified and spotted in an equimolar ratio on the Mouse Exonic Evidence Based Oligonucleotide (MEEBO) array. Images were preview scanned on an Axon 4000B and channels were balanced until an overall Cy5/Cy3 ratio of 1 was achieved before a final data scan at 5µm was performed in GenePix. The images were manually gridded, and then saved for analysis. Data were analysed using Spotfire Decision Site. Gene Ontology terms were downloaded from BioMart <http://www.biomart.org> version NCBI37, 09 April 2009. Genes with Biological Process term "regulation of transcription" (GO:0045449) were assumed to encode transcription factors. Of 276 probes with an absolute log2 ratio >2, 231 probes were present in BioMart; the remaining cDNA probes did not match currently known genes.

Project description:The goal of this study was to examine differences in gene expression of tumor specific CD8 T cells in an in vivo tumor mouse model after inhibition of galectin-3 protein expression by genetic knockout. Galectin-3 is thought to modulate CD8 T cell response by cross-linking cell surface glycoproteins Galectin-3 is a 31 kD carbohydrate-binding lectin that is over-expressed by many human malignancies. It also modulates T cell responses through a diverse array of mechanisms including induction of apoptosis, TCR cross linking in CD8+ T cells, and T cell receptor (TCR) down regulation in CD4+ T cells. We found that patients responding to a granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting allogeneic pancreatic tumor vaccine developed post immunization antibody responses to galectin-3 on a proteomic screen. We used the HER-2/neu (neu-N) transgenic mouse model to study galectin-3 binding on adoptively transferred high avidity neu-specific CD8+ T cells derived from TCR transgenic mice. Here, we show that galectin-3 binds preferentially to activated antigen-committed CD8+ T cells only in the tumor microenvironment (TME). Galectin-3 deficient mice exhibit improved CD8+ T cell effector function and increased expression of several inflammatory genes when compared with wild type (WT) mice. We also show that galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3 mediated suppression of CD8+ T cells in vitro. Lastly, galectin-3 deficient mice have significantly elevated levels of circulating plasmacytoid dendritic cells (pDCs), which are superior to conventional dendritic cells (cDCs) in activating CD8+ T cells. Binding of galectin-3 to cell-surface glycoproteins on immune cells suppresses a pro-inflammatory immune response. Thus, inhibiting galectin-3 in conjunction with CD8+ T cell directed immunotherapies should enhance the tumor specific immune response. 3 different experimental groups were studied. Galectin-3 WT CD8 T cells adoptively transferred into Galectin-3 WT mice, galectin-3 WT CD8 T cells transferred into galectin-3 KO mice, and finally galectin-3 KO CD8 T cells transferred into galectin-3 KO mice. Galectin-3 WT CD8 T cells transferred into Galectin-3 WT mice were used as the reference group. Four biological replicates were submitted for each group, and adoptively transfered CD8 T cells were isolated 5 days post-adoptive transfer into tumor-bearing mice treated with a whole cell GM-CSF secreting vaccine. Cells were purified by cell sorting on the Thy1.2 surface marker.

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Immediate dysfunction of vaccine-elicited CD8+ T cells primed in the absence of CD4+ T cells

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