Dr. Mario Roederer’s expert team studies single cells to better characterize immunology systems involved in the treatment of HIV. The team uses non-human primate models to advance T-cell characterization methods for single-cell gene expression profiling. To perform their research, they are using state-of-the-art technologies, including 18-color FACS systems and the Fluidigm Biomark System for single-cell gene expression. Basic research interests include:
Roles and interactions of the components of the mature central immune system
- Correlates of pathogenesis
Understanding how acute infection occurs in tandem with massive infection and destruction of the CD4 memory cells Mario Roederer
- Immunological correlates of protection for vaccine development
Studying the effects of prior vaccination in preventing CD4 memory cell destruction, and the function of lymphocytes in preventing viral proliferation
- Advances and development of cytometric technology
Utilizing state-of-the-art instrumentation including FACS systems and Fluidigm instruments, reagents, analysis, and assays
Dr. Roederer and his team have a methods paper entitled, “Highly multiplexed quantitation of gene expression on single cells” (Journal of Immunological Methods, in press 2013).
“Comparatively little work has been performed in immunological settings, where great cellular heterogeneity is already known.”
— Mario Roederer, Ph.D.
In the paper, Roederer and his team propose a method to interrogate single T-cells utilizing multiplexed TaqMan assays on the Biomark platform. Initially, T-cells were index sorted using the BD FACSAria™ to obtain single cells. Next, Roederer details his methodology to ensure that assays performed optimally on bulk RNA prior to profiling the single cells. Roederer, et al, demonstrated the limit of detection (LOD) as low as a single mRNA transcript. This showed that the Biomark has high sensitivity and wide dynamic range to detect single-cell equivalents of mRNA.
The team monitored T-cell activation at the single-cell level, identifying subsets of CD4+ cells within the population. In fact, CD4+ cells showed that co-expression of gene combinations like CSCR5/CCL5 and DPPR/TIA1 were rare events, rather than common occurrences previously observed in bulk mRNA.
Heterogeneity was further established when Staphlyococcal enterotoxin B (SEB)-response was compared between CD154+ and CD154- T-cells. When stimulated, analysis demonstrated a subset of genes strongly associated with T-cell activation. However, only 5-20% of T-cells possess the receptor Vβ chain variant to bind SEB.
In this study, cellular heterogeneity is demonstrated to have strong immunological relevance. Mario Roederer and his team continue to advance systematic, single-cell methods to better understand cellular and molecular interactions of the central immune system.