(425f) Finding Murine t-Cell Receptor Repertoire Shifts Due to Ovalbumin Challenges Using High Throughput Sequencing

Thymocyte driven cellular immunity utilize membrane-bound receptors to recognize specific disease antigens and initiate the signaling cascade to provide the appropriate immune response. An individual may have billions of thymocyte cells (t-cells) each with a unique receptor comprise their repertoire and with newly developed high throughput sequencing (HTS) technologies these expressed protein receptors can be sequenced. Analyzing the shifts in t-cell receptor (TCR) repertoires due to disease challenges can elucidate how immune systems respond and adapt to cues from the environment. The TCR is formed from specific chromosomal regions surrounding a highly variable, antigen interrogating portion of the receptor. Challenging an individual with a particular antigen known to give a specific immune response is thought to shift or expand the repertoire to include new TCRs directly targeting the foreign disease antigen. These responses are observed as changes in the distribution and diversity of TCR sequences when compared with repertoires in healthy and unchallenged individuals.

HTS of the TCR mRNA harvested from sorted CD4⁺ & CD8⁺ t-cells shows the conserved nature of endogenous and exogenous antigen responses and how the immune system selects different t-cell lines to combat diseases and antigens. Examination of the TCR-gene recombinations, VJ-regions in the α-chain and VDJ-regions of the more complex β-chain, gives insight into the mechanisms of t-cell maturation and selection process. This can also explain evolutionarily how the modern immune system has developed whether through co-evolution with common diseases and viruses or through other mechanisms.

Preliminary experimental data show proper recognition of utilized genes and recombination sequence with distinct TCR repertoires existing in the control groups and cell lines. Presently, error rates in the 454 sequencing reactions and t-cell gene recognition recover approximately 40% of the all TCR specific sequences harvested from lymph node t-cells. Developing better mRNA isolation techniques and sequence analysis algorithms to increase recognition accuracy and anticipate erroneous miscalled nucleotides will increase the total number of functional sequences collected in the experiment and create a platform technology for analyzing and characterizing the immune system on an individual basis.