1. Deep sequencing of the human TCRγ and TCRβ repertoires provides evidence that TCRβ rearranges after αβ, γδ T cell commitment C.S. Carlson 1, A. Sherwood 2, C. Desmarais 2, R.J. Livingston 2, J. Andriesen 2, M. Haussler 3, H. Robins 1 1 Fred Hutchinson Cancer Research Center, 2 Adaptive TCR Corporation 3 University of Manchester Deep sequencing of the human TCRγ and TCRβ repertoires provides evidence that TCRβ rearranges after αβ, γδ T cell commitment C.S. Carlson 1, A. Sherwood 2, C. Desmarais 2, R.J. Livingston 2, J. Andriesen 2, M. Haussler 3, H. Robins 1 1 Fred Hutchinson Cancer Research Center, 2 Adaptive TCR Corporation 3 University of Manchester For additional information about immunoSEQ assays and the immunoSEQ Analyzer suite of bioninformatics applications at Adaptive TCR Technologies, visit our booth or contact us on the web at www.adaptivetcr.com and www.immunoseq.com.www.adaptivetcr.com www.immunoseq.com This work is published in Science Translational Medicine, July 2011, Vol. 3, Issue 90. Adaptive TCR Technologies Suite 300 307 Westlake Ave N Seattle, WA 98109 For additional information about immunoSEQ assays and the immunoSEQ Analyzer suite of bioninformatics applications at Adaptive TCR Technologies, visit our booth or contact us on the web at www.adaptivetcr.com and www.immunoseq.com.www.adaptivetcr.com www.immunoseq.com This work is published in Science Translational Medicine, July 2011, Vol. 3, Issue 90. Adaptive TCR Technologies Suite 300 307 Westlake Ave N Seattle, WA 98109 Results  The most frequent TCRG nucleotide clone in each individuals is public and shared by all three healthy individuals (Fig. 4). Fig. 4: Shared nucleotide identical TCRγ CDR3 sequences: Nine nucleotide identical TCRγ CDR3 sequences amplified from γδ T cells are shared by all three individuals. For each shared sequence, the copy count detected for each individual is indicated on the Y-axis. Conclusions 1.The TCR  CDR3 region rearranges prior to T cell differentiation (Table 1, Fig. 5). 2.The TCR  CDR3 region rearranges after T cell commitment (Table 2, Fig. 5). 3.The TCR  CDR3 repertoire is clonal (Fig. 2A), and >70% of chains carried by  T cells use V  9-J  P1 gene segments (Fig. 3A). 4.The highest frequency TCR  CDR3 sequence in each individual is public and shared by all 3 subjects (Fig. 4). Fig 5: TCR CDR3 rearrangement schema Results  The most frequent TCRG nucleotide clone in each individuals is public and shared by all three healthy individuals (Fig. 4). Fig. 4: Shared nucleotide identical TCRγ CDR3 sequences: Nine nucleotide identical TCRγ CDR3 sequences amplified from γδ T cells are shared by all three individuals. For each shared sequence, the copy count detected for each individual is indicated on the Y-axis. Conclusions 1.The TCR  CDR3 region rearranges prior to T cell differentiation (Table 1, Fig. 5). 2.The TCR  CDR3 region rearranges after T cell commitment (Table 2, Fig. 5). 3.The TCR  CDR3 repertoire is clonal (Fig. 2A), and >70% of chains carried by  T cells use V  9-J  P1 gene segments (Fig. 3A). 4.The highest frequency TCR  CDR3 sequence in each individual is public and shared by all 3 subjects (Fig. 4). Fig 5: TCR CDR3 rearrangement schema Introduction The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino- acid sequence of the hypervariable complementary determining region 3 (CDR3) regions of the T cell receptor (TCR). The genes that encode the two primary types of TCRs,  and , undergo somatic rearrangement during T cell development. TCRβ and TCRδ genes are assembled via recombination of Variable (V), Diversity (D), and Joining (J) gene segments (VDJ recombination) and similarly, the TCR  and TCR  genes by recombination of Variable and Joining gene segments (VJ recombination) to form productive  and  Y-like surface receptors. During development, the TCR variable regions do not rearrange simultaneously in the multi- potent precursor T cell; TCR  rearranges first, followed by TCR  and TCR . The TCR  locus rearranges last, after the surface expression of both pre-TCR  and TCR  chains(18). Both the order and the effect of TCR rearrangement and expression on  and  T-cell lineage commitment remains controversial (1, 19-23). The canonical model proposes that TCR , TCR , and TCR  rearrange prior to T cell lineage commitment. Adaptive TCR has developed a method to deeply sequence both TCRB and TCRG CDR3 chains. By sequencing the TCRB and TCRG repertoire of both types of T cells, we will be able to estimate:18119-23 1. Abundance of rearranged TCR  CDR3 chains in  T cells. 2. Abundance of rearranged TCR  CDR3 chains in  T cells 3. Clonality of the TCR  and TCR  repertoire. 4. Overlap of  T cell TCRG CDR3 repertoire between any two individuals. Materials and Methods  40 ml of whole blood collected from three healthy adult donors.  PBMC isolated with Ficoll gradient and bead-sorted using Miltenyi kits to isolate and collect αβ and  T cells.  Genomic DNA extracted from sorted cells with Qiagen DNAeasy macro-kit  TCR  and TCR  sequences amplified and sequenced from both  and  T cells using the immunoSEQ assay (Fig. 1) Fig. 1: TCRB Assay Introduction The ability of T lymphocytes to mount an immune response against a diverse array of pathogens is primarily conveyed by the amino- acid sequence of the hypervariable complementary determining region 3 (CDR3) regions of the T cell receptor (TCR). The genes that encode the two primary types of TCRs,  and , undergo somatic rearrangement during T cell development. TCRβ and TCRδ genes are assembled via recombination of Variable (V), Diversity (D), and Joining (J) gene segments (VDJ recombination) and similarly, the TCR  and TCR  genes by recombination of Variable and Joining gene segments (VJ recombination) to form productive  and  Y-like surface receptors. During development, the TCR variable regions do not rearrange simultaneously in the multi- potent precursor T cell; TCR  rearranges first, followed by TCR  and TCR . The TCR  locus rearranges last, after the surface expression of both pre-TCR  and TCR  chains(18). Both the order and the effect of TCR rearrangement and expression on  and  T-cell lineage commitment remains controversial (1, 19-23). The canonical model proposes that TCR , TCR , and TCR  rearrange prior to T cell lineage commitment. Adaptive TCR has developed a method to deeply sequence both TCRB and TCRG CDR3 chains. By sequencing the TCRB and TCRG repertoire of both types of T cells, we will be able to estimate:18119-23 1. Abundance of rearranged TCR  CDR3 chains in  T cells. 2. Abundance of rearranged TCR  CDR3 chains in  T cells 3. Clonality of the TCR  and TCR  repertoire. 4. Overlap of  T cell TCRG CDR3 repertoire between any two individuals. Materials and Methods  40 ml of whole blood collected from three healthy adult donors.  PBMC isolated with Ficoll gradient and bead-sorted using Miltenyi kits to isolate and collect αβ and  T cells.  Genomic DNA extracted from sorted cells with Qiagen DNAeasy macro-kit  TCR  and TCR  sequences amplified and sequenced from both  and  T cells using the immunoSEQ assay (Fig. 1) Fig. 1: TCRB Assay Results  Utilization of V  -J  gene segment pairs is non-random. The V  9-JP  1 gene segment pair is observed much more often relative to other V  -J  gene segment pairs (Fig. 3A).  Utilization of specific V β and J β segments is variable within an individual, but relatively consistent between individuals (Fig. 3C). Fig. 3: Average V-J gene utilization of sequenced TCRγ and TCRβ sequences across three samples: Average V-J utilization of gene segments in TCRγ CDR3 sequences amplified from γδ T cells (3A), TCRγ CDR3 sequences amplified from αβ T cells (3B), and TCRβ sequences amplified from αβ T cells (3C). 3B ), and TCRβ sequences amplified from αβ T cells (3C). Results  Utilization of V  -J  gene segment pairs is non-random. The V  9-JP  1 gene segment pair is observed much more often relative to other V  -J  gene segment pairs (Fig. 3A).  Utilization of specific V β and J β segments is variable within an individual, but relatively consistent between individuals (Fig. 3C). Fig. 3: Average V-J gene utilization of sequenced TCRγ and TCRβ sequences across three samples: Average V-J utilization of gene segments in TCRγ CDR3 sequences amplified from γδ T cells (3A), TCRγ CDR3 sequences amplified from αβ T cells (3B), and TCRβ sequences amplified from αβ T cells (3C). 3B ), and TCRβ sequences amplified from αβ T cells (3C). Results  Both  and  T cells carry rearranged TCR  CDR3 chains (Table 1). Table 1. Summary of total and unique TCR  Sequences   T cells carry few to no rearranged TCR  CDR3 chains (Table 2). Table 2: Summary of total and unique TCR  Sequences  For all three individuals, the  T cell TCR  repertoire is dominated by one or two clones (>50% of total repertoire) (Fig. 2). Fig. 2: Frequency of the 25 most common TCR sequences: For each sample we plot the proportion of productive sequences accounted for by the 25 most numerous productive TCR sequences. (2A) TCRγ chains amplified from γδ T cells and αβ T cells and (2B) TCRβ chains amplified from αβ T cells. Results  Both  and  T cells carry rearranged TCR  CDR3 chains (Table 1). Table 1. Summary of total and unique TCR  Sequences   T cells carry few to no rearranged TCR  CDR3 chains (Table 2). Table 2: Summary of total and unique TCR  Sequences  For all three individuals, the  T cell TCR  repertoire is dominated by one or two clones (>50% of total repertoire) (Fig. 2). Fig. 2: Frequency of the 25 most common TCR sequences: For each sample we plot the proportion of productive sequences accounted for by the 25 most numerous productive TCR sequences. (2A) TCRγ chains amplified from γδ T cells and αβ T cells and (2B) TCRβ chains amplified from αβ T cells. A B C