Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis of gene expression of Th1 and Tfh LCMV-specific Smarta CD4 T cell populations

ABSTRACT: In this study, we sought to distinguish phenotypically and functionally distinct virus-specific effector CD4 T cell subsets that form during acute lymphocytic choriomeningitis virus (LCMV) infection and to examine their ability to develop into memory T cells. To further characterize the effector subsets and identify genetic pathways and transcription factors involved in their differentiation, we performed genome-wide gene expression profiling of the three day 8 effector cell populations: (1) PSGL1hi Ly6Chi, (2) PSGL1hi Ly6Clo and (3) PSGL1lo Ly6Clo Smarta CD4 T cells along with (4) day 60 memory PSGL1hi and (5) naïve Smarta CD4 T cells using Illumina BeadChips. Results of this microarray confirmed the validity of our phenotyping by flow cytometry in many ways and showed distinct gene signatures for the three effector subsets. DNA microarray analysis was performed on two or three independent samples of five different Smarta CD4 T cell populations: (1) naïSGL1lo, (3) day 8 PSGL1hi Ly6Clo, (4) day 8 PSGL1hi Ly6Clo and (5) day 60 PSGL1hi memory cells.

ORGANISM(S): Mus musculus

SUBMITTER: Hailong Meng

PROVIDER: E-GEOD-32596 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity. Fluorescence-activated cell sorting (FACS) was used to sort subsets of CD4 T cells from blood (peripheral blood mononuclear cells; PBMC) and lymph node from a cohort of HIV-infected people with natural control of the virus (termed HIV controllers). Subsets of CD4 T cells that were sorted are as follows: from blood, (1) naÃ¯ve (N), (2) central memory (CM), (3) transitional memory (TM), and (4) effector memory (EM); from lymph node, (1) naÃ¯ve (N), (2) non-germinal center T-follicular helpers (nGC), (3) germinal center T-follicular helpers (Tfh), (4) effector memory (EM), and (5) other central memory-like subsets (CMPD1lo57lo, CMPD1lo57hi, and/or CMPD1lo). Total RNA and total DNA were extracted from these sorted subsets in separate fractions using RNAzol RT and DNAzol. Total cellular DNA was used for HIV quantification and sequence analysis as describe in the publication. Total RNA used for mRNA library construction by oligo-dT purification (Dynabeads), random fragmentation by heating in the presence of Magnesium (in form of 5x first-strand buffer, LifeTech), reverse transcription (SuperScript III), and then second-strand synthesis, end repair, a-tailing, and adaptor ligation using NEBNext enzyme master mixes and oligonucleotides. Libraries were sequenced on an Illumina HiSeq2000. Please note that each 'source name' value represent individual who havs HIV infection with natural control of the virus.

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Genome-wide analysis of gene expression of Th1 and Tfh LCMV-specific Smarta CD4 T cell populations

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