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Deep profiling of the mouse TCRb CDR3 region in thymus and spleen Cindy Desmarais1, Chris Carlson1,2, Harlan Robins1,2, Joe Blattman3, and Robert Livingston1 Adaptive TCR Technologies, Seattle, WA1, Fred Hutchinson Cancer Research Center, Seattle, WA2, Department of Immunology, University of Washington, Seattle, WA3 Introduction To recognize a diverse and unpredictable universe of antigens, the adaptive immune system generates a remarkable breadth of diversity by combinatoric shuffling of T cell receptor (TCR) gene segments in somatic cells. The TCR signals an immune response by the lymphocyte when the TCR binds to an antigen displayed on the MHC of an infected cell. The TCR is a heterodimer composed of two chains, either the αβ or the γδ, the expression of which is determined during maturation of the naive T cell in the thymus. Prior to exiting the thymus, the developmental pathway includes a number of selection events that commits the T cell to express either the αβ or the γδ lineage by productively rearranging the TCR gene segments. The αβ-committed T cells then undergo additional selection events to screen out the thymocytes in which the TCR recognizes its cognate MHC ligand too avidly, thereby removing potential self-reacting T cells in a process termed negative selection. Conversely, during positive selection thymocyte survival and its developmental lineage are determined by the ability of the TCR to bind the MHC complex. Results Ultra-deep sequencing of the mouse TCRb CDR3 regions in lymphocytes from spleen and thymus generated 17.1 million and 9.0 million sequences, respectively. The highest copy number CDR3 sequence is shared between thymus and spleen and is a public TCRb clonotype, identified seven times in the NCBI nonredundant database of protein sequences. The size of the naive mouse αβ repertoire is estimated to be approximate two million clones of approximately 10 cells each (Casrouge et al, J Immunol 164:5782-5787, 2000). The theoretical maximum diversity of 1015 unique αβ TCR (Davis and Bjorkman, Nature 334:395-402, 1988) far exceeds the observed repertoire illustrating the combined effects of positive and negative selection to narrow the set of functional TCRs. To profile the diversity of murine TCR we used massively parallel DNA sequencing with highly specialized software tools to generate and analyze millions of TCRβ sequences from naïve T cells in the thymus and mature T cells of spleen. Methods To profile and compare the diversity of the murine TCRβ repertoire of thymus and spleen, we used ultrahigh-throughput DNA sequencing with a proprietary multiplex PCR methodology to generate tens of millions of TCRβ sequences. Total genomic DNA was extracted from the spleen and thymus of an adult female C57BL/6 mouse and subjected to the immunoSEQ TCRβ multiplex PCR assay targeting the CDR3 region of the mouse TCRβ locus. Millions of TCRβ sequences were generated following a PCR amplification consisting of 36 forward V segment and 14 reverse J segment primers which targeted all possible somatic combinations of the rearranged TCRβ cell receptor locus in 3.2 micrograms of the genomic DNA, equivalent to approximately 106 haploid genomes. The forward and reverse PCR primers contain on the 5’ ends, the universal sequences compatible with the Illumina solid phase amplification. Following purification by silica solid phase extraction, the TCRβ CDR3 PCR library was quantified and loaded on an Illumina flow cell for sequencing on an Illumina Genome Analyzer GAIIx. Sequencing primers were anchored in a conserved J segment motif and used to collect 60 bases of sequence, sufficient to uniquely identify the V, D and J segments and span the length of the CDR3 region. Conclusions Ultra-deep sequencing generated 5.8 million thymus and 11.7 million spleen TCRb CDR3 sequences from one million input genomes demonstrating greater than five-fold sampling of the input genomes. The immunoSEQ mouse TCRb assay can detect functional selection among millions of CDR3 sequences as demonstrated by the increased high frequency sequences and shorter CDR3 lengths in the spleen compared to the thymus. Comparison of the distinct CDR3 sequence copy numbers between thymus and spleen revealed more high frequency sequences in spleen. The average productive spleen CDR3 sequence was shorter by 1.8 nucleotides compared to the average productive thymus sequence, consistent with prior observations suggesting positive selection by MHC, demonstrating the ability of this assay to detect functional selection among millions of T cell receptor sequences. This length difference was not observed in the non-productive CDR3 sequences. Raw GA sequences were pre-processed to remove errors in the primary sequence and to compress the data. Each sequence was required to have a six nucleotide match to one of the 36 V and one of the 14 J segments, as previously defined by the ImMunoGeneTics collaboration. The CDR3 region was defined as the amino acid sequence from the second conserved cysteine at the 3’ end of the V segment to the conserved phenylalanine in the J segment. The total number of nucleotides between these codons determines the length and therefore the reading frame of the CDR3 region. Processed sequence data were deposited in the immunoSEQ secure proprietary relational database management system. Data analysis for the visualization, sorting, selection, and comparison of the TCRβ sequences was performed using the immunoSEQ Analyzer suite of bioinformatics software programs available at www.immumoseq.com. Analysis of CDR3 sequences identified a high frequency CDR3 sequence shared between thymus and spleen and represented a public TCR clonotype. Future directions Develop additional immunoSEQ assays and analysis tools targeting the mouse TCRa, TCRd, TCRg, and IGH loci to profile these components of the murine immune system. Assemble a database of public TCRb clonotypes observed in various mouse strains and environments as a resource for the mouse immunology community. For additional information about immunoSEQ assays and the immunoSEQ Analyzer suite of bioinformatics applications at Adaptive TCR Technologies, visit our booth or contact us on the web at www.adaptivetcr.com and www.immunoseq.com. Adaptive TCR Technologies Suite 300 307 Westlake Ave N Seattle, WA 98109 Apply the immunoSEQ mouse TCRb assay to explore further questions of thymic selection and other applications of the mouse model such as vaccine development, dysfunction of the adaptive immune system, disease resistance, transplantation and immune reconstitution.