Deep sequencing of the human TCRγ and TCRβ repertoires provides evidence that TCRβ rearranges

after αβ, γδ T cell commitmentC.S. Carlson1, A. Sherwood2, C. Desmarais2, R.J. Livingston2, J. Andriesen2, M. Haussler3, H. Robins1

1Fred Hutchinson Cancer Research Center, 2Adaptive 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. This work is published in Science Translational Medicine,

July 2011, Vol. 3, Issue 90.

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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 TCRg CDR3 region rearranges prior to T cell differentiation (Table 1, Fig. 5).

2. The TCRb CDR3 region rearranges after T cell commitment (Table 2, Fig. 5).

3. The TCRg CDR3 repertoire is clonal (Fig. 2A), and >70% of chains carried by gd T cells use Vg9-JgP1 gene segments (Fig. 3A).

4. 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

IntroductionThe 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:

1. Abundance of rearranged TCRg CDR3 chains in ab T cells.

2. Abundance of rearranged TCRb CDR3 chains in gd T cells

3. Clonality of the TCRg and TCRb repertoire.

4. Overlap of gd 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 gd T cells.

Genomic DNA extracted from sorted cells with Qiagen DNAeasy macro-kit

TCRb and TCRg sequences amplified and sequenced from both ab and gd T cells using the immunoSEQ assay (Fig. 1)

Fig. 1: TCRB Assay

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

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 gd T 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.

A

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