Deep sequencing of the human TCRγ and TCRβ r epertoires provides evidence that TCRβ rearranges after αβ, γδ T cel

1. Deep sequencing of the human TCRγ and TCRβ repertoires provides evidence that TCRβ rearranges after αβ, γδT cell commitment C.S. Carlson1, A. Sherwood2, C. Desmarais2, R.J. Livingston2, J. Andriesen2, M. Haussler3, H. Robins1 1Fred Hutchinson Cancer Research Center, 2Adaptive TCR Corporation 3University of Manchester • 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, andTCR 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: • Abundance of rearranged TCRg CDR3 chains in ab T cells. • Abundance of rearrangedTCRb CDR3 chains in gdT cells • Clonality of the TCRg and TCRb repertoire. • Overlap of gdT 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 gd T cells. • Genomic DNA extracted from sorted cells with QiagenDNAeasy macro-kit • TCRb and TCRg sequences amplified and sequenced from both aband gdT cells using the immunoSEQ assay (Fig. 1) • Fig. 1: TCRB Assay • Results • Both ab and gd T cells carry rearranged TCRg CDR3 chains (Table 1). • Table 1. Summary of total and unique TCRg Sequences • gd T cells carry few to no rearranged TCRb CDR3 chains (Table 2). • Table 2: Summary of total and unique TCRb Sequences • For all three individuals, the gdT cell TCRg 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 • Utilization of Vg-Jg gene segment pairs is non-random. The Vg9-JPg1 gene segment pair is observed much more often relative to other Vg-Jg gene segment pairs (Fig. 3A). • Utilization of specific Vβ and Jβ 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 • 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 • The TCRg CDR3 region rearranges prior to T cell differentiation (Table 1, Fig. 5). • The TCRb CDR3 region rearranges after T cell commitment (Table 2, Fig. 5). • The TCRg CDR3 repertoire is clonal (Fig. 2A), and >70% of chains carried by gd T cells use Vg9-JgP1 gene segments (Fig. 3A). • The highest frequency TCRg CDR3 sequence in each individual is public and shared by all 3 subjects (Fig. 4). • Fig 5: TCR CDR3 rearrangement schema A B C 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. 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