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

摘要

Thymocyte driven cellular immunity utilize membrane-bound receptors to recognize specificdisease antigens and initiate the signaling cascade to provide the appropriate immune response. Anindividual may have billions of thymocyte cells (t-cells) each with a unique receptor comprise theirrepertoire and with newly developed high throughput sequencing (HTS) technologies theseexpressed 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 cuesfrom the environment. The TCR is formed from specific chromosomal regions surrounding a highlyvariable, antigen interrogating portion of the receptor. Challenging an individual with a particularantigen known to give a specific immune response is thought to shift or expand the repertoire toinclude new TCRs directly targeting the foreign disease antigen. These responses are observed aschanges in the distribution and diversity of TCR sequences when compared with repertoires inhealthy and unchallenged individuals.HTS of the TCR mRNA harvested from sorted CD4~+ & CD8~+ t-cells shows the conservednature of endogenous and exogenous antigen responses and how the immune system selects differentt-cell lines to combat diseases and antigens. Examination of the TCR-gene recombinations, Vjregionsin the α-chain and VDJ-regions of the more complex β-chain, gives insight into themechanisms of t-cell maturation and selection process. This can also explain evolutionarily how themodern immune system has developed whether through co-evolution with common diseases andviruses or through other mechanisms.Preliminary experimental data show proper recognition of utilized genes and recombinationsequence with distinct TCR repertoires existing in the control groups and cell lines. Presently, errorrates in the 454 sequencing reactions and t-cell gene recognition recover approximately 40% of theall TCR specific sequences harvested from lymph node t-cells. Developing better mRNA isolationtechniques and sequence analysis algorithms to increase recognition accuracy and anticipateerroneous miscalled nucleotides will increase the total number of functional sequences collected inthe experiment and create a platform technology for analyzing and characterizing the immune systemon an individual basis.