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
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
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.
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.
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
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 and
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.

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