Transcriptomic Analysis of CD8 T cells from HIV+ Patients Using Fluidigm Technology

Introduction:  Our lab is using the Fluidigm BioMark System to analyze gene expression patterns in primary immune cells.  The Fluidigm technology uses microfluidics to perform high-throughput multiplex RT-PCR on multiple samples.  In this study we evaluated gene expression using a panel of 96 specific Taqman primer/probe assays on purified CD8 T cells from eight treatment-naïve HIV infected patients participating in a clinical trial to study the effect of Raltegravir intensification with combination anti-retroviral therapy.  Methods:  At entry and 48 weeks post-treatment we activated CD8 T cells isolated from PBMC with anti-CD3 and anti-CD28 antibodies for 48 hours.  cDNA was synthesized from harvested RNA and the samples were run on the Fluidigm 96.96 dynamic array chip.  Cycle threshold values were obtained for bioinformatic analyses in which we analyzed differentially expressed genes and fold change expression in activated vs. unactivated cells at both time points.  Results:  The number of differentially expressed genes between unactivated and activated CD8 T cells was different at entry and 48 weeks post-treatment.  Six genes were significantly induced at both time points: CD25, IL-2, IL-4, DUSP4, IL12Rb2 and BCL6B. These genes were the only induced genes at entry however, following treatment a total of 24 genes were significantly induced.  Further, the induced genes that were shared in both time points displayed higher fold changes at 48 weeks compared to entry.   Conclusion:  Combination anti-retroviral therapy (48 weeks) drastically altered CD8 T cell responses from HIV infected individuals making cells more responsive to stimulation.  These findings confirm the observation that CD8 T cells from HIV infected people exhibit an ‘exhausted’ phenotype.