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The somatically generated T cell receptor CDR3α contributes to the MHC allele specificity of the T cell receptor

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Mature T cells bearing αβ T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor β chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and β chain and, surprisingly, dramatically affected by the non germ line encoded CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.
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The somatically generated T cell receptor CDR3 contributes to the MHC allele specificity
of the T cell receptor
Philippa Marrack,1,2,3 Sai Harsha Krovi,3 Daniel Silberman,2,3 Janice White,2 Eleanora
Kushnir,2 Maki Nakayama,3,6 James Crook,4 Thomas Danhorn,4 Sonia Leach,2,4 Randy
Anselment,4 James Scott-Browne,5 Laurent Gapin,3 and John Kappler,1,2,3
1Howard Hughes Medical Institute, Denver, CO 80206;
2Department of Biomedical Research, National Jewish Health, Denver, CO 80206;
3Department of Immunology and Microbiology, University of Colorado School of Medicine,
Aurora, CO 80045;
4Division of Biostatistics and Bioinformatics, National Jewish Health, Denver, Colorado 80206
5La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037;
6Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine,
Aurora, CO 80045.
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ABSTRACT
Mature T cells bearing  T cell receptors react with foreign antigens bound to alleles of major
histocompatibility complex proteins (MHC) that they were exposed to during their development
in the thymus, a phenomenon known as positive selection. The structural basis for positive
selection has long been debated. Here, using mice expressing one of two different T cell
receptor chains and various MHC alleles, we show that positive selection-induced MHC bias
of T cell receptors is affected both by the germline encoded elements of the T cell receptor
and chain and, surprisingly, dramatically affected by the non germ line encoded CDR3 of the
T cell receptor chain. Thus, in addition to determining specificity for antigen, the non germline
encoded elements of T cell receptors may help the proteins cope with the extremely
polymorphic nature of major histocompatibility complex products within the species.
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INTRODUCTION
Many T lymphocytes in the body express clonally distributed T cell antigen receptors composed
of alpha and beta chains (TCRs) that react with peptides derived from pathogens bound in a
groove on the surface of host major histocompatibility proteins (MHCs). The genes encoding
these MHC proteins are the most polymorphic genes in a given species. Most of the
polymorphisms tend to be concentrated within the residues that line the peptide-binding groove
of the molecules. Hence, in general, different MHC alleles within a species preferentially bind,
and present to TCRs, different peptides from any given invading organism. Thus the pathogen
is unlikely to mutate such that none of its peptides bind to any of the MHC proteins expressed
within the target species and the immune responses of at least some individuals within the
infected species will thus be able to deal with the invading pathogen.
Many years ago another consequence of MHC polymorphisms was recognized. The allelic
variants of MHC expressed in one individual are very frequently recognized by 1% or more of
the T cells of other individuals expressing different MHC alleles, a phenomenon called
“alloreactivity”. While differences in bound peptides play an important role in alloreactivity(Hunt
et al., 1990, Crumpacker et al., 1992), structural studies show that some of the allelic variations
in MHC proteins themselves interact with the TCRs of alloreactive T cells (Grandea and Bevan,
1993, Archbold et al., 2008, Colf et al., 2007).
Experiments have shown that T cells in one individual are more likely to react with foreign
peptides bound to the grooves of self MHC than to foreign peptides bound to foreign MHC (Fink
and Bevan, 1978, Zinkernagel et al., 1978, Kappler and Marrack, 1978, Sprent, 1978). This
phenomenon, known as positive selection, is caused by the fact that thymocytes are allowed to
develop into mature T cells only if the TCR they bear reacts with low affinity/avidity with MHC
proteins bound to self peptides in the thymus (Sprent et al., 1988, Ashton-Rickardt et al., 1994,
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Sebzda et al., 1994, Hogquist et al., 1994). Paradoxically, in an apoptotic process termed
“negative selection”, the thymus generally weeds out T cell progenitors that react with too high
affinity/avidity with self MHC plus self peptide, thus preventing the maturation of many
potentially self reactive T cells (Kappler et al., 1987, von Boehmer et al., 1989). Thus the
collection of TCRs on mature T cells in any individual bears the footprint of positive selection,
reacting almost undetectably with self MHC bound to self peptide and being more likely to react
with foreign peptides bound to alleles of MHC to which they were exposed in the thymus than to
peptides bound to unfamiliar MHC (Fink and Bevan, 1978, Zinkernagel et al., 1978, Kappler and
Marrack, 1978, Sprent, 1978, Hunig and Bevan, 1981).
Mutational and structural studies have shown that the alpha and beta chains that comprise
TCRs each usually engage MHC + peptide via three complementary determining loops (CDRs
1,2 and 3) (Garcia et al., 1996, Reinherz et al., 1999, Dai et al., 2008). For both the TCR alpha
and beta chains, two of these loops, CDR1 and CDR2, are encoded by the germ line TRAV (for
the TCR chain) and TRBV (for the TCR chain) genes. The third, CDR3, loop for each chain,
on the other hand, is produced during TCR gene rearrangement as the cells develop in the
thymus (Davis, 1985). Thus, the sequence coding for CDR3 ,for example, is created when
one of many TRAV gene segments rearranges to fuse with one of the many TRAJ gene
segments with the total number of possible CDR3 sequences increased by removal and/or
addition of bases at the joining points of TRAV and TRAJ (Gellert, 2002, Cabaniols et al., 2001,
Moshous et al., 2001, Lu et al., 2008). This process creates the DNA coding for the entire V
domain. The stretch of DNA coding for CDR3 is constructed along the same lines, by joining of
one of a number of TRBV, TRBD and TRBJ gene segments, again with bases removed or
introduced at the joining points to form the CDR3 loop of the complete V domain.
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T cell receptors react with a surface of MHC plus peptide that is made up of amino acids from
two MHC alpha helices that flank the groove where the peptide is bound. In the dozens of
solved structures of TCRs bound to peptide-MHC complexes a pattern has emerged in which,
usually, the germ line encoded TRAV and TRBV CDR1 and CDR2 loops focus on the MHC
alpha helices whereas the somatically generated TCR CDR3 loops focus on the peptide (Garcia
et al., 1999, Reinherz et al., 1999, Colf et al., 2007, Rudolph et al., 2006). Thus one might
predict that positive selection selects TCRs that react well with peptides bound to self rather
than foreign MHC by picking out TCRs bearing TRAVs and TRBVs that react favorably with self
MHC. Indeed there is evidence that this is the case (Pircher et al., 1992, Merkenschlager et al.,
1994, Sim et al., 1996). However, different MHC alleles will present different self peptides to
developing thymocytes, therefore it is also possible that it is the presented peptide rather than
the MHC protein itself, that governs the allele bias of positive selection. Again some evidence
suggest that the selecting peptide is crucial (Ignatowicz et al., 1996, Tourne et al., 1997, Nikolic-
Zugic and Bevan, 1990, Hogquist et al., 1994, Ashton-Rickardt et al., 1994, Wong and
Rudensky, 1996, Barton et al., 2002), results that favor the idea that CDR3 sequences dominate
positive selection. Understanding of this issue is complicated by the cooperative nature of TCR
interactions with its ligands, by which an interaction at one site on the TCR/MHC/peptide
surface adjusts interactions elsewhere (Mazza et al., 2007, Baker et al., 2012, Adams et al.,
2016) and a study that indicated that the entire sequence of the TCR chain, including the
TRAV, TRAJ and CDR3, is involved in positive selection (Merkenschlager et al., 1994).
We set out to resolve these issues. We analyzed the TCR repertoires of naïve CD4 T cells in
mice that each expressed one of two TCR chains, DOWT or DO48A (Scott-Browne et al.,
2009), and a single MHCII protein, IA, of alleles b, f or s (for simplicity and ease of reading we
will use IA to describe what are often termed I-A proteins, and we will not use superscripts to
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denote MHC and IA alleles). As predicted by previous studies (Pircher et al., 1992,
Merkenschlager et al., 1994, Sim et al., 1996), the frequency with which mature T cells used
different TRAVs was indeed affected to some extent by the MHCII allele on which they were
positively selected and by the coexpressed TCR. Likewise the TRAJs used were affected by
the selecting MHCII allele and coexpressed TCR, but demonstrated unexpected biases
towards use of the TRAJs that were furthest from the TRAV locus.
Most surprisingly, however, the CDR3 sequences differed markedly depending on the MHCII
allele and partner TCR in the mouse. This was true even if we compared, between MHC
alleles, the TCR sequences constructed from rearrangements involving the same TRAVs and
TRAJs, indicating that the non germ line encoded portions of CDR3are involved in MHCII
allele specific selection.
RESULTS
The generation and properties of mice expressing a single TCR beta chain
The impact of positive selection on the TCR repertoire of mature T cells cannot be understood
by sequencing only the expressed TCR or TCR chains. This is because others and we have
found a fairly high percentage of individual TCR or TCR sequences are expressed in animals
regardless of their MHC haplotype (Robins et al., 2010, Warren et al., 2011, Liu et al., 2014)
(Supplementary Table 1). Presumably this is at least in part possible because each individual
chain is paired with a different partner(s) in animals with different MHC alleles. Therefore, the
pairs of TCR chains expressed in individual T cells must be known in order to understand the
impact of thymus selection on the TCR repertoire of mature T cells.
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The T cells in any given mouse or human have been reported to bear, collectively, more than
105 different TCRand about the same number of different TCR chain sequences (Venturi et
al., 2011, Li et al., 2016). Thus the T cells might bear up to 1010 different combinations of these
chains. Although methods for sequencing and accurately pairing the TCR and TCR (or
immunoglobulin heavy and light chain) RNAs from many individual T (or B) cells have been
described (Tan et al., 2014, DeKosky et al., 2013), in our experience (Munson et al., 2016)
these are still not able to cope with the large numbers of individual chains and combinations we
expect in normal animals. Therefore we decided to limit our analyses to the T cells in animals
that expressed a single TCR and any possible TCR. This choice has two advantages. It
allowed accurate knowledge of the TCR on the T cells and, because it was expected that only
a limited number of TCR chains can be positively selected with a single TCR, it limited the
numbers of different TCRsequences we expected to find in the mice (Merkenschlager et al.,
1994, Fukui et al., 1998, Hsieh et al., 2006).
We chose two TCR chains for these experiments (Supplementary Table 2). These were the
TCR originally isolated from a T cell hybridoma constructed from BALB/c T cells specific for IAd
or IAb bound to a peptide from chicken ovalbumin, the DOWT TCR (White et al., 1983) and
the same TCR with a mutation in its TRBV region such that the tyrosine at position 48 was
changed to an alanine, DO48A (Scott-Browne et al., 2009). This mutation reduces the ability
of the TCR chain to react with the alpha chain alpha helix of MHCII and with the alpha1 alpha
helix of MHCI. The chain was chosen for our analyses because we thought that the TCR
sequences that could successfully overcome the deficits in MHC recognition by the TCR chain
might more clearly illustrate the properties of the TCR needed for successful positive selection.
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The goal of these studies was to find out how the allele of MHC involved in thymic selection
affects the sequences of the TCRs on the selected T cells. To achieve this we studied TCRs on
naïve CD4 T cells that had been selected in some of the readily available mice that expressed a
single MHCII protein, IAb, IAf and IAs (Mathis et al., 1983). Transgenic mice that expressed
either DOWT or DO48A and no other TCR were crossed such that they each expressed one
of these MHCII alleles. The numbers of mature CD4 and CD8 T cells in the thymuses of the H2
b, f or s strains of mice were measured. As predicted by our previous data using retrogenic
mice (Scott-Browne et al., 2009), the numbers of mature CD4 or CD8 thymocytes in mice
expressing DO48A were much lower than those in mice expressing DOWT (Fig. 1A). This
was true regardless of the MHC allele on which the cells were selected. Thus the TCR 48A for
48Y substitution affects MHC interactions regardless of the MHC class or allele, as we have
previously predicted (Scott-Browne et al., 2009). The difference in numbers of mature T cells
between mice expressing DOWT and DO48A was less marked in peripheral lymph nodes
than in the thymus (Fig.1B), probably because of increased homeostatic expansion, as
exemplified by the increased percentages of CD44hi T cells amongst those few that could
mature in DO48A mice (Fig. 1C).
There were more mature CD4 than CD8 T cells in the lymph nodes of mice expressing DOWT
and H2b (Fig 1D). The effect was much less marked in mice expressing H2f or H2s. The
phenomenon may be due to the fact that DOWT was found in a TCR that reacts with IAd or
IAb plus a foreign peptide (OVA 327-339) and not from an MHCI-reactive TCR (White et al.,
1983). The bias towards CD4 versus CD8 T cells in H2b animals was not manifest in lymph
node cells bearing DO48A and was, indeed, reversed in animals expressing that TCR and
H2f or H2s.
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The TCR confers a bias towards reactivity with the selecting MHC allele
Mature T cells do not usually react detectably with self MHC alleles plus self peptides, the
reactivity that presumably allowed their positive selection in the thymus. However, the potential
inadequacies of DO48A allowed us to test whether or not the TCRs that, on mature T cells,
paired with it did indeed react preferentially with the MHCII allele on which they were positively
selected. We guessed that introduction of the more prominently MHC-reactive DOWT chain
into DO48A T cells might reveal the underlying reactivity of the TCR sequences in these T
cells for various MHC alleles. Thus we isolated CD4 T cells from mice expressing the DO48A
transgene, stimulated them with anti-TCR, transduced them with a GFP+ retrovirus expressing
the DOWT chain, and tested the ability of the transductants to react with cells expressing
different alleles of MHC. CD69 expression was used as a marker of activation. Non transduced
(GFP-) cells in the same cultures were used as controls. In no case did the nontransduced cells
show a significant response. However, some of the DOWT transduced cells responded.
Notably, the percentage of the transduced cells that responded to challenge was always
greatest if the antigen presenting cells expressed the MHC allele on which the T cells were
positively selected (Fig. 2). For example, T cells from a DO48A H2b mouse, after transduction
to express DOWT, were most likely to react with H2b presenting cells and DOWT transduced
cells from DO48A H2s mice reacted only with challenge cells expressing H2s. These
experiments show that the TCR chain that pairs with the transgenic DO48A does indeed
contribute to the preference of CD4 T cells to react with peptides bound to the MHCII allele
involved in positive selection.
Expression of only one TCR chain limits the numbers of TCR sequences that can
participate in positive selection
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Because allelic exclusion of the TCR locus is not perfect (Malissen et al., 1992), mature T cells
may express two functional TCR proteins. To be sure that the TCR chains analyzed in our
experimental mice were actually those involved in positive selection of the cells bearing them,
we crossed the DOWT or DO48A transgenic, TCR-/- mice with TCR-/- TCR-/- animals of
each MHC haplotype to generate animals that were DOWT or DO48A transgenic, TCR-/-,
TCR+/-. Naïve CD4 T cells were isolated from the lymph nodes of these animals and cDNA
coding for their TCRs were sequenced as previously described (Silberman et al., 2016). PCR
and sequencing errors in the germ line encoded portions of these sequences were corrected as
described in the Materials and Methods section. To deal with possible sequencing errors in the
non germ line encoded portions of CDR3, sequences that occurred only once in any given
sequencing run were eliminated from further analysis. In fact this decision affected the
conclusions of all the experiments show below only slightly. Conclusions from analyses that
included all sequences, or that eliminated sequences that occurred with the lowest 5%
frequency in each sample were similar (data not shown).
Others have previously reported that the T cells in mice expressing a single TCR chain have a
limited repertoire of TCR chains by comparison with WT animals (Fukui et al., 1998). To find
out whether this applied to CD4 T cells expressing the DOWT or DO48A TCR we
constructed species accumulation curves for TCR sequences in B6 and the TCR transgenic
animals. These were performed by combining the TCR sequences from all mice of the same
genotype (Fig. 3) or by plotting the TCR sequences for individual mice of each genotype
(Supplementary Fig. 1). Species accumulation curves show that the total number of TCR
sequences we could detect on naïve CD4 T cells in the TCR transgenic animals ranged from
less than 5,000 to a maximum of about 30,000. These numbers are less than those found in
CD4 naïve T cells from B6 mice, which we found to be similar in number to those found on
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mouse CD8 T cells (Genolet et al., 2012), >than 105 in number (Fig. 3). The numbers of TCR
sequences that could partner with DOWT in selection of CD4 T cells varied considerably with
the selecting MHCII allele. More than 4 times more TCR sequences were apparent in mice
expressing IAb versus IAs (Fig. 3B-D), perhaps because the TCR from which DOWT is
derived can be selected by IAb (Liu et al., 1996). This effect of MHC allele on the numbers of
selected TCR chains was not evident in animals expressing DO48A. Notably, the numbers of
different TCR chains associated with DO48A was lower than those associated with DOWT
regardless of the MHCII allele involved, possibly because of the extra demands imposed on
TCR chains by the inadequate TCR chain lacking an important MHC contact residue, Y48
(Scott-Browne et al., 2009).
Perhaps the real surprise in these results is how many TCR sequences can partner with a
single TCR and participate successfully in positive selection since work in humans and mice
suggest that, on peripheral T cells, each TCR partners with only about 5-25 different TCRs
(Arstila et al., 1999, Casrouge et al., 2000, Venturi et al., 2011, Li et al., 2016).
Expressed TCR sequences are strongly influenced by the selecting MHC allele and
partner TCR.
We compared the frequency with which particular TRAV/TRAJ/CDR3 amino acid sequences,
i.e. the entire TCR sequences, occurred in the various strains of mice. Data of this type can
be compared in several ways. The data can be analyzed to find out whether a particular
TRAV/TRAJ/CDR3 sequence occurs in each sample, regardless of how often it appears in the
set (comparison of unique sequence use). In this case, Jaccard similarity coefficients can be
used to measure the similarity between samples. On the other hand, use of particular
TRAV/TRAJ/CDR3 sequences can be compared taking into account the number of times a
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particular combination occurs. In this case Anne Chao Jaccard abundance based indices
(Chao et al., 2012) are an appropriate statistical tool. Both methods were used in the
comparisons shown in Fig. 4. Jaccard analyses showed that the same combination of
TRAV/TRAJ/CDR3 sequences were likely to appear in samples from mice of the same TCR
and MHC genotype but were very unlikely to be shared with the T cells from mice expressing a
different MHC allele (Fig. 4A). This was just as apparent when the abundance with which the
sequences were expressed was taken into account (Fig. 4B). Thus these data show that, given
a single TCR, the TCR sequences that can participate in positive selection are dramatically
affected by the selecting MHCII allele. Moreover, the fact that the values of the Anne Chao
Jaccard analyses for mice of the same MHCII allele are much larger than those of the Jaccard
analyses shows that sequences that appear frequently in one mouse of a given genotype are
more likely to be found in other mice of the same type. Such a result is a manifestation of the
fact that some sequences were repeated many times in all mice of a given MHCII type, whereas
other sequences were rare. This uneven and certainly non-Poissonian distribution of TCR
sequences has been observed before (Correia-Neves et al., 2001, Fazilleau et al., 2005,
Freeman et al., 2009). The phenomenon was not necessarily caused by expansion of single
clones of T cells since, even in sets in which the CDR3 amino acid sequences were identical,
the DNA sequences were not necessarily all the same (data not shown).
Similar analyses were applied to samples from mice in which the selecting MHCII allele was
identical, but the partner TCR differed. The data in Figs. 4C and D show that the selected
TCR chains depended on the partner TCR, even when the selecting MHC allele was
identical. Close inspection revealed, however, that there was slightly more overlap if the co-
selected TCRs were different but the selecting MHCII alleles were identical than if the reverse
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were true, that is the co-selected TCRs were the same but the selecting MHCII alleles were
different (Supplementary Fig 2) (Fink and Bevan, 1978).
A few sequences appear in at least one mouse of each haplotype. For example, 16 sequences
appear in DOWT mice expressing MHCII b, f or s (but not in any DO48A animals) (data not
shown). Such sequences might belong to yet undiscovered types of T cells that express an
invariant TCR, like iNKT cells or MAIT cells(Chandra and Kronenberg, 2015, Gapin, 2009).
We think this is unlikely to be true because, in the complete naïve CD4 T cell sequences, we did
not consistently find the sequences of the iNKT cell or MAIT cell TCRs. Probably this was
because the cells bearing the iNKT cell or MAIT cell invariant TCRs were in the
activated/memory T cell populations, which were not examined in our experiments. Were there
to be an undiscovered T cell subset bearing another invariant TCR it would presumably also
be in the activated/memory T cell population and therefore not included in our assays.
These data show that positive selection acts on CD4 T cell precursors, via the action of the
expressed MHCII allele on particular TCR/TCR pairs.
TRAV usage depends on the selecting MHCII haplotype and the partner TCR chain.
In order to find out which element(s) of TCR determine MHC allele specificity we analyzed
each element separately using data from the experiments described above. Others have
previously reported that certain TRAVs are used more frequently by CD4 versus CD8 T cells or
in mice expressing particular alleles of MHC (Jameson et al., 1990, Pircher et al., 1992, Sim et
al., 1996, Simone et al., 1997, Merkenschlager et al., 1994). TRAV rearrangements occur in
thymocytes after the cells have rearranged their TCR genes (Lindsten et al., 1987). Thus, the
frequency with which TRAVs appear on mature naïve CD4 T cells is predicted to depend on a
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number of issues, the ease with which the TRAV gene can rearrange (Chen et al., 2015), its
ability to pair with the preexisting TCR expressed in the cell (Vacchio et al., 1993) and the
ability of the TCR/TCR pair to participate in positive, but not negative, selection on the MHCII
protein expressed in the thymus.
We first tested whether the expressed MHC haplotype might unexpectedly affect the nature of
TRAVs expressed on preselection thymocytes. As shown in Fig. 5A and Supplementary Fig
3, TRAV usage on preselection thymocytes was similar, regardless of the MHC allele in the
donor animal or the coexpressed TCR(s). There were, however, some interesting aspects of
TRAV use on preselection thymocytes (Supplementary Fig 3). TRAVs whose genes are most
proximal to the TRAJ locus (TRAVs 17-21) were frequently rearranged, as predicted by
previous studies (Villey et al., 1996, Shih et al., 2011, Genolet et al., 2012) (Supplementary Fig
4) (note that the TRAVs are arranged by family and not by position in the TRAV locus).
However, in preselection thymocytes we also observed frequent rearrangements involving
TRAV 1 and members of the TRAV 3, 6, 7, 10, 11 and 14 families. In the cases of the TRAV
families the frequently rearranged TRAVs were not always those which are most proximal to the
TRAJ locus. For example, amongst the TRAV7 family, the most frequently rearranged member
was TRAV7-2D, one of the family members that is furthest from the TRAJ locus, whereas the
close relative of TRAV7-2D, TRAV7-2A, was not frequently rearranged. This suggests that
chromatin structure, promoter accessibility and use by rearranging processes also play a role in
TRAV rearrangements (Chen et al., 2015).
TRAV use by T cells from mice of the same genotype was very similar (Fig. 5 B-G,
Supplementary Fig. 4 and 5). However, the selecting MHC allele affected the frequency with
which different TRAVs were expressed on mature naïve CD4 T cells (Fig 5 and
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Supplementary Figs. 4 and 5). For example members of the TRAV5 family were used to
some extent by CD4 T cells selected on IAb, but not by cells selected on IAf or IAs (compare
Figs. 5B, C with Figs. 5D-G). On the other hand, CD4 T cells in DOWT H2s mice were alone
in their use of TRAV17 (Fig 5 B, D and F). Differential use of TRAVs was much more marked
in DO48A mice and illustrated the TRAV preferences of mice selected on different MHCII
alleles more strikingly. For example, DOWT cells selected on IAs used most members of the
TRAV6 family whereas DO48A expressing cells selected on the same MHC allele used, of the
TRAV 6 family, almost entirely TRAV6-5D and TRAV6-7DN and also used more frequently than
T cells selected on other MHCII alleles, members of the TRAV16 family. Perhaps this reflects a
greater need for basic amino acids in TRAV CDR1 and CDR2 for selection of H2s with DO48A
as a partner, since, of the TRAV6 family, TRAVs6-5D and TRAV6-7DN (and TRAV6-5A and
TRAV6-7DN) have a total of two basic amino acids in these elements whereas other members
of the family have none. Likewise all expressed members of the TRAV16 family contain 2 or 3
basic amino acids in their CDR1 and CDR2 segments. This narrowing in TRAV choice by
DO48A cells may reflect the increasing demands for selection imposed by the absence of the
tyrosine at position 48 of the TCR chain.
Our analyses are based on a method in which cDNAs from individual mouse T cells are
amplified simultaneously with a reverse TRAC oligo and oligos built to match each TRAV family
(see Methods Section). Therefore the differences in TRAV discovery could be due to more
efficient PCR amplification of some TRAV genes than others. However, since the efficiencies of
detection will be similar between members of the same family, it is legitimate to compare the
frequency of rearrangement between different members of the same family, or the frequency of
use of the same TRAV in mature T cells selected on different MHCs or with different TCR
partners (see below).
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TRAJ usage depends on the selecting MHCII haplotype and the partner TCR chain.
TRAJ use by preselection thymocytes was similar regardless of the selecting MHC haplotype or
co-expressed TCR (Fig. 6A). TRAJ use by naïve CD4 T cells from B6 mice was fairly uniform
across the locus (Fig. 6B). Unexpectedly, however, and in contrast to preselection thymocytes
and naïve CD4 T cells from B6 mice, TRAJ use by T cells from mice expressing a single TCR
was much more uneven and tended towards TRAJs whose genes were distal to the TRAV locus
( Fig. 6 B-L). Regardless of MHC allele, CD4 T cells in DOWT animals used TRAJ21 most
frequently (Fig. 6 D-F). The reasons for this bias are unknown. TRAJ21 contains a tyrosine at
or near the contact point with MHC but other TRAJs have a tyrosine similarly situated and they
are not overexpressed. Moreover TRAJ21 is not overexpressed in T cells expressing DO48A,
T cells that might be expected to be even more readily selected with an added tyrosine (Scott-
Browne et al., 2009). The bias towards use of distal TRAJ genes was even more marked in
animals expressing DO48A. In these mice TRAJ 9, TRAJ 12 and TRAJs 9,15 and 31
dominated in H2b, H2f and H2s mice respectively (Fig. 6 G-I). Pairwise comparisons between
different mice are shown in Supplementary Fig. 6 and DESeq 2 analyses are in
Supplementary Fig 7.
We do not know why the distal TRAJ genes were preferred in mice in which the TCR
repertoire was limited by the presence of a single TCR. In another study with a fixed TCR
chain, a bias towards proximal TRAJs was noted with TRAV17, a TRAV that is close to the
TRAJ locus (Casanova et al., 1991). The same publication described biases, depending on
MHC allele towards use of Type 1 (G rich) or type 2 TRAJs. These explanations don’t apply
here, since in the experiments presented here TRAV expression was not particularly biased
towards the distal TRAVs and the used TRAJs don’t fall particularly into Types 1 or 2. It is
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possible that the choice is related to the DO chain itself. Alternatively it maybe that, because it
is difficult for thymocytes expressing a single TCR to find a TCRthat can pair with the TCR
and contribute to positive selection, multiple TCR rearrangements have to occur in each
thymocyte before a suitable TCR partner is found. This will inevitably drive expressed TCRs
towards use of the distal TRAJs, although why these should satisfy the demands of positive
selection more frequently than the proximal TRAJs do, is not obvious, at least from their amino
acid sequences. It has recently been reported that prolonged expression of RAG protects cells,
to some extent, from death (Karo et al., 2014). If the thymocytes in TCR transgenic mice have
to express RAG for a longer time to find a suitable TCR partner, then the prolonged expression
of RAG needed for the multiple rearrangements required to access the TRAC proximal TRAJs
might preferentially allow survival of the thymocytes in which this prolonged expression has
occurred. Preliminary analyses of the naïve CD4 T cells in the various mice did not, however,
suggest that the T cells in the TCR transgenic mice were more resistant to death than the
equivalent cells in B6 animals.
Thus overall, like the use of TRAVs, use of TRAJs depended on both the MHC haplotype and
TCR present in the animals.
Expressed CDR3 sequences are strongly influenced by the selecting MHC allele and
partner TCR.
Because of the removal or introduction of bases when TRAVs rearrange to TRAJs, CDR3
protein sequences can vary in the number of amino acids they encode between the conserved
C terminal cysteine of the TRAV and the conserved phenyl alanine/leucine/tryptophan glycine
pair in the TRAJ region. We compared the average lengths of CDR3s and the predicted
number of N region bases between mice expressing the same TCR and different MHCII
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alleles. CD4 T cells selected on IAb had significantly shorter CDR3 lengths and fewer N
region bases than their counterparts selected on IAf or IAs (Supplementary Fig. 8).
The analyses shown in Fig 4 compared the frequencies with which entire TCR sequences
appeared under different selecting circumstances. We also analyzed how often particular
CDR3 sequences are found in mice that differed in the selecting MHC allele or in the co-
expressed TCR, using, again, Jaccard or Anne Chao Jaccard analyses to compare particular
sequences without or with taking into account the abundance with which they occurred. Data
comparing the occurrence of CDR3 protein sequences between mice that expressed the same
or different MHCII alleles are shown in Fig. 7A and B, and data comparing the occurrence of
CDR3 protein sequences between mice expressing the same MHCII but different partner
TCRs are shown in Fig. 7C and D. The results were similar to those obtained when
comparing the entire TCR sequences. The expressed CDR3 sequences in mice with a
particular MHCII allele were very unlikely to be found in CD4 T cells of mice expressing a
different MHCII allele, even if the co-selected TCR were the same in the mice (Figs 7A and
B). Likewise, CDR3 sequences co-selected with a particular TCR were unlikely to be shared
with those co-selected with a different TCR, even if the selecting MHCII allele were the same.
The first few amino acids of CDR3 (defined as the stretch between the last C of the TRAVs
and the conserved F/W/L G sequence of the TRAJs) are encoded by the TRAVs themselves.
Likewise, the last few amino acids of CDR3 are encoded by the TRAJs. Therefore the fact
that the CDR3 sequences are controlled by the MHCII allele on which they were selected
might have been, to some extent, dictated not by the non germline encoded amino acids in
CDR3 but rather by the TRAV encoded amino acids downstream of the cysteine at the C
terminal end of the TRAVs or by the TRAJ encoded amino acids upstream of their conserved
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TRAJ F/W/L. This problem applies particularly to the use of TRAVs since TRAV CDR1 and
CDR2 amino acids may contact MHCII and thereby contribute to thymic selection whilst also
dictating the first few amino acids of the accompanying CDR3 region.
We therefore checked whether CDR3 sequences associated with particular TRAV/TRAJ pairs
differed between T cells selected on different MHCII alleles or associated with different TCRs.
Only a few of the possible TRAV/TRAJ pairs were present in sufficient numbers in all of the
mice to be compared, so only a few such comparisons could be made. Examples of such
comparisons are shown in Fig. 8. Summaries combining all allowable results (in which all the
TRAV.TRAJ combinations to be compared included least 5 different CDR3 sequences/mouse)
are shown in Supplementary Fig. 9. T cells expressing DOWT and the same TRAV and
TRAJ combinations, but selected on different MHCII alleles or with different TCRs clearly had
CDR3 sequences that were almost completely unique to the selecting MHCII alleles.
A recent study has reported that thymocytes with aromatic/hydrophobic amino acids at the tips
of their CDR3 segments are biased towards MHC reactivity, regardless of the selecting MHCII
allele(Stadinski et al., 2016). The observations in the paper applied to CD4+ CD8+ (double
positive thymocytes) that had been positively, but not negatively, selected, identified by their
expression of CD69, and to regulatory T cells compared to preselection thymocytes. Such cells
have not been examined in the experiments described here, so we cannot tell directly whether a
similar observation applies to TCR sequences. On the whole the evidence is that cells with
aromatic amino acids at positions 6 and 7 on CDR3 are not particularly eliminated by clonal
deletion in the thymus (data not shown). Nevertheless, we evaluated individual amino acids
that would probably be at the tips of CDR3s in CDR3 of different lengths. The results show
MHCII allele and TCR specific selection for particular amino acids and also changes in amino
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acid preference at CDR3 positions depending on the length of the CDR3 (Supplementary
Figure 10). For example, arginine was very frequently used at position 4 in 12 amino acid long
CDR3s selected by IAs with DO48A, and similarly over selected at position 5 in 14 amino
acid long CDR3s selected on IAf with DOWT, but much less frequently used by other MHC
selection, TCR, CDR3 length combinations. Phenyl alanine was only used with evident
frequency at position 5 in 14 amino acid-long CDR3s selected on IAb with DOWT. Apart
from the phenyl alanine result there was no particular enrichment for aromatic amino acids at
these tips.
Discussion
It has long been known that T cells bearing TCRs are biased towards recognition of antigenic
peptides bound to the allele of MHC to which the T cells were exposed in the thymus(Fink and
Bevan, 1978, Zinkernagel et al., 1978, Kappler and Marrack, 1978, Sprent, 1978). This
phenomenon, known as positive selection, has been ascribed to a requirement for a low
affinity/avidity reaction between the developing thymocyte and MHC proteins to which the cell is
exposed in the thymus cortex(Sprent et al., 1988). However, the peptides presented to
immature T cells in the thymus are also controlled by the allele of MHC involved. Thus, the
MHC allele specificity of positive selection might be dictated by TCR contact with the MHC-
engaged peptides rather than the MHC protein itself. If this were the case, positive selection
might be dominated by the portion of TCRs that most consistently engages the peptides bound
to MHC, CDR3 sequences of TCRs, rather than the germ line encoded TRAVs and TRBVs.
This idea is supported by the fact that, in some cases, peptides related to the activating antigen
can stimulate positive selection of thymocytes bearing particular TCRs (Ashton-Rickardt et al.,
1994, Sebzda et al., 1994, Hogquist et al., 1994, Kraj et al., 2001, Smyth et al., 1998). Moreover
MHC proteins that were supposed to differ only in amino acids that bind peptide and that don’t
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contact TCRs nevertheless were found to differ in their ability to select thymocytes bearing
certain TCRs (Nikolic-Zugic and Bevan, 1990).
During positive selection could TCRs detect allelic differences between MHCs directly?
Although that MHC amino acids that contact TCRs are quite well conserved (Bjorkman et al.,
1987) they do vary somewhat between alleles (Reche and Reinherz, 2003). For the MHCII
alleles studied here, the amino acids pointing towards the TCR are, at most positions, uniform,
but IAb differs from IAf and IAs with a two amino acid insertion on the surface of its beta chain
alpha helix, an insertion that causes an allele specific bulge in this helix. IAs also differs from
the other MHCII alleles we studied with alpha chain H68Y and beta chain R70Q changes and
position 72 of the MHCII alpha helix is a V in IAb, an I in IAf and IAs. Therefore the solvent
exposed residues on the alpha helices of the MHCII proteins themselves could contribute to
allele specific positive selection. These amino acids are contact points, not only for the CDR1
and CDR2 loops of TCRs, but also, sometimes for the TCR CDR3 regions. For example, in the
structure on a TCR bound to the complex of IAu bound to a myelin basic protein, CDR3
engages polymorphic amino acids at positions 55 and 81 of the IAu alpha chain and CDR3
engages polymorphic amino acids at positions 65 of the IAu alpha chain and positions 67 and 70
of the IAu beta chain (Maynard et al., 2005).
The findings presented previously (Merkenschlager et al., 1994) and here, demonstrate that the
allele of MHCII involved in positive selection affects the frequencies with which TRAV and
TRAJ elements are selected and, most dramatically, the CDR3 sequences that appear on
mature T cells, as previously indicated for CD8 T cells (Ferreira et al., 2006). The N terminal
portion of CDR3 is provided by the TRAV, so the effects of MHCII on TRAV choice by a
particular TCR could actually be due to demands placed on the CDR3 rather than on the
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TRAV itself. On the other hand, since the CDR3 sequence is affected in part by its co-
expressed TRAV, the demands of positive selection could be entirely on the TRAV, not the
CDR3. We think it likely that all three of the TCR CDRs can play a role in positive selection.
However, clearly CDR3 is involved since the sequences in the center of this element vary
depend on the selecting MHCII allele, even if the accompanying TRAV and TRAJ are the same
(Fig. 8).
Overall, the results strongly suggest that positive selection allele specificity involves recognition
of both MHC and peptide (reviewed in (Klein et al., 2014, Vrisekoop et al., 2014). In fact, given
the geometry with which TCRs engage their MHC/peptide ligands, it is difficult to imagine that
this would not be the case.
The data here also show that the sequence of the TCR chain affects the TCR and CDR3
that can participate in positive selection almost as much as the selecting MHC allele does. Not
only does the TCR affect which TCRs and which CDR3s will be successful, it also
determines how many different TCRs can do the job since, regardless of the MHCII allele
involved, fewer TCRs can be selected with DO48A than with DOWT. These results are
similar to those observed earlier that showed that fewer CD4 T cells are selected in DO48A-
expressing versus DOWT-expressing mice. Together these suggest that DO lacking an
important MHC contact amino acid, the “Y” at position 48, places more stringent requirements
on TCR for successful thymus selection (Scott-Browne et al., 2009).
Overall, the results show that the entire TCR sequence plays a role in positive selection. How
can this be, given that selection is thought to occur during low affinity reactions? Naively one
might have predicted that relatively few TCR-to-MHC/peptide interactions would be needed to
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reach the needed energy of interaction and these could be provided by just a portion of the
TCR, not the entire molecule as suggested here. Some TCR configurations may interfere with
contact with MHC/peptide or prevent the proper engagement of CD4 or CD8. Other TCR
configurations may react too strongly with their ligand, leading to negative selection. This idea
may apply to up to 70% of all TCRs (Ignatowicz et al., 1996, Stritesky et al., 2013). Competition
for selecting ligands may also play a role. Also to be bourne in mind is the fact that here we are
observing the consequences of many TCR selection events, some TCRs may be selected
based on their TRAVs, others via their TCRs, with the observed results showing biases by
both of these. Nevertheless the detrimental effects of an inappropriate CDR3 cannot be
overcome by other elements of TCR.
There are problems with the notion that the bound peptide is a determinant of MHC allele
specific positive selection. Most notably, the fact that mature T cells, after selection on a single
MHCII allele bound to a single peptide can respond to peptides that are unrelated in sequence
to the selecting peptide (Pawlowski et al., 1996, Ignatowicz et al., 1997, Nakano et al., 1997,
Ebert et al., 2009, Lo et al., 2009). Moreover, teleologically, the idea that the selecting peptides
in the thymus are the only feature that governs T cell specificity doesn’t seem evolutionarily
favorable. Such might limit the ability of T cells to respond to foreign peptides that are unrelated
to those in the thymus. Nevertheless, self peptides might provide an advantage anyway, by
supporting the survival of mature T cells and also, perhaps, T cell responses to unrelated
peptides when the self and foreign peptides are presented on the same cells (Kirberg et al.,
1997, Wulfing et al., 2002). However, MHC-bound peptides on thymus cortical epithelial cells
are not necessarily the same as those on peripheral cells (Honey et al., 2002, Murata et al.,
2007) so this advantage may not be available for all T cells.
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In the studies presented here, the total number of TCRs that can be selected with a single
TCR ranges between about 4,600 and 30,000, depending on the selecting MHCII allele and
partner TCR (Fukui et al., 1998). Are these numbers surprisingly low or high? Based on the
estimated numbers of TCRs and TCRs that appear in the periphery of an individual, it has
previously been estimated that each TCR chain can be successfully selected with up to 25
different TCRs (Arstila et al., 1999, Casrouge et al., 2000). Yet here, and in previous studies, it
appears that, for a single TCR, the number of possible TCR partners is at least 3 orders of
magnitude larger. How to account for this large disparity? Probably it is caused by competition
for selection in the thymus, a phenomenon that has been previously demonstrated (Martins et
al., 2014, Visan et al., 2006). In a wild type thymus, each of the immature thymocytes is
competing with a huge number of others bearing disparate TCR and TCR sequences. In the
thymus of a mouse expressing a single TCR, the immature thymocytes bear the same large
number of TCRs, and now all those expressing an even approximately suitable TCR have
the opportunity to be positively selected. This idea may be related to the profound bias towards
distal TRAJs reported here, and therefore a predicted increased time available for
rearrangements for the thymocytes in single TCR transgenic animals.
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Materials and Methods
Mice
Mice were purchased from the Jackson Laboratory, Bar Harbor ME and subsequently interbred
in the Biological Research Center at National Jewish. Plasmids coding for the DO11.10 TCR
chain (DOWT) or its mutant, in which the tyrosine at position 48 was replaced by an alanine
(DO48A) were created, with the human CD2 promoter to drive expression of the genes (White
et al., 1983, Greaves et al., 1989). DNAs coding for the promoters and genes were injected into
fertilized C57BL/6J (B6) eggs at the Mouse Genetic Core Facility at National Jewish Health.
Mice produced from these eggs were crossed with animals lacking functional TCR genes
(Mombaerts et al., 1991) and with B10.M (H2f) or B10.S (H2s) animals to create animals
expressing the transgenic TCR genes, no other TCRgenes and H2b, f or s. By similar
intercrosses animals were produced that expressed no functional TCR or TCR genes and
H2b, f or s. These animals were intercrossed to give rise to animals expressing either DOWT
or DO48A, no other TCR genes, TCR+/- and H2 b, f or s. Animals were subsequently used
for analysis if they expressed the TCR locus derived from B6 rather than B10 animals.
Animals were handled in strict accordance with good animal practice as defined by the relevant
national and/or local animal welfare bodies, and all animal work was approved by the National
Jewish Health Animal Care and Use Committee (IACUC). The protocol was approved by
National Jewish IACUC (protocol number AS2517).
T cell isolation
Cells were isolated from the thymuses, spleens (B6 analyses) or peripheral lymph nodes
(DOWT or DO48A analyses) of 6-14 week old mice. CD4 T cells were isolated by negative
selection on MACS columns (Milltenyi Biotech). The cells were stained with antibodies
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conjugated to a fluorochrome and specific for: Pacific Blue-CD4, Alexa488-TCR, PE-B220, PE-
TCR, PE-CD8, PE-Cy5-CD25, Alexa647-CD44, PE-Cy7-CD62L, purchased from BD
Pharmingen or eBioscience or produced in house. The cells were sorted based on their
expression of CD4, TCR, low levels of CD44 and high levels of CD62L and absence of staining
with PE and PE-Cy5. Cells were sorted into staining buffer (BSS, 2% fetal bovine serum, plus
sodium azide) by a MoFloXDP (Beckman Coulter Life Sciences or Synergy SY3200 (iCyt)
instruments at the National Jewish Health Flow Cytometry Core Facility.
Retroviral infection of T cells.
Retroviruses expressing DOWT or DO48A and green fluorescent protein were produced as
described in (Scott-Browne et al., 2009). CD4 T cells were purified, by negative selection on
Automax columns, from the spleens and lymph nodes of DO48A transgenic, TCR-/- mice
expressing various MHC alleles. The cells were activated by 24h culture on plates pre-coated
with anti-TCR (Ham597) and anti-CD28 (37.51) The supernatants were then removed from
the plates and replaced with supernatants containing the DOWT or DO48A retroviruses and
8ug/ml polybrene in culture medium. The cells were spun at 2000G in bags containing
10%CO2/90%air at 37.C for 2h. At this point the medium was replaced with complete culture
medium containing 10% fetal bovine serum and cultured for 1d followed by addition of IL-2.
Three days later the cells were harvested and challenged as described below.
Assessment of MHC reactivity of transduced T cells
Red blood cell depleted spleen cells from mice expressing various MHC alleles were cultured
overnight with IL-4 plus GM-CSF. The cells were then thoroughly washed and used, at a dose
of 106 cells/well, to stimulate 106/well TCR transduced CD4+ T cells, prepared as described
above. These wells were cultured for 51/2 hours in a final total volume/well of 200ul of CTM.
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The cells were then fixed (Permafix), stained and analyzed for expression of CD4 (PerCP anti-
CD4), GFP and CD69 (PE anti-CD69).
TCR sequencing and analysis
RNA was isolated from purified naïve CD4 T cells, PCR’d to expand TCRa sequences and
sequenced as described in (Silberman et al., 2016). Post-sequencing analysis was performed
to identify the TRAV and TRAJ genes for each sequence along with its corresponding CDR3.
TRAV family and subfamily members were assigned based on the IMGT designations with
modifications based on our own analysis of expressed TRAV sequences in B6 mice. IMGT has
identified two gene duplication events in the B6 TRAV locus, the “original” genes, most of which
are closest to the TRAJ locus are designated by their family number and a number indicating
their subfamily membership. Here, for ease of analysis, we have added the letter “A” to their
designation, eg TRAV01-1A. TRAV subfamily members in the IMGT designated duplicated “D”
and new “N” genes we add the letters “D” or “N”, eg TRAV07-6D or TRAV07-6N. In some
cases the entire nucleotide sequences of subfamily members are identical and, therefore,
indistinguishable by our analyses. In these cases the subfamily members are designated to
include all possible source genes, eg TRAV06-3ADN or TRAV06-6AD.
Errors occur during sequencing reactions and accumulate as the numbers of sequences
acquired increase (Bolotin et al., 2012, Liu et al., 2014). The sequences were all corrected for
errors in the TRAV and TRAJ elements, which do not somatically mutate. However, because
the amino acids in and flanking the non germ line encoded CDR3 regions could not be
corrected, sequences with errors in these elements are bound to appear at some low frequency
and cause a gradual rise in the species accumulation curves. To eliminate these misreads we
decided to include in our analyses only those TCRsequences that occurred more than once in
each sample. To correct for sequencing errors within the CDR3, the sequences were modified
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by replacing erroneous nucleotides with the appropriate germline-encoded nucleotides
whenever a discrepancy was observed. Such correction was possible only when a nucleotide
difference could be resolved by aligning to the germline TRAV and/or TRAJ genes. To avoid
making inappropriate changes to the potentially non germline encoded portions of CDR3, such
corrections were applied only if the change from the germline sequence occurred more than 3
nucleotides before the predicted end of the TRAV genes or more than three nucleotides after
the predicted end of the TRAJ gene. Finally, the amino acid usage within the CDR3 was
determined for each sequence to identify any patterns in the CDR3 regions in sequences
belonging to T cells from one MHC haplotype versus another. All of the analysis was performed
using in-house programs developed in Python 2.7.
In order to represent the differential TRAV and TRAJ gene usage in TCRs sequenced from
different mouse samples, we used edgeR from the R/Bioconductor package. A threshold of p <
0.05 was used to identify genes that were most significantly differentially expressed between
samples.
Euclidean distances for TRAVs and TRAJs were calculated as log2 transformed counts per 104
sequences.
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Acknowledgements
The authors thank Dean Becker for production of the transgenic mice, Desiree Garcia and
Tabitha Turco for help in mouse breeding, Shirley Sobus and Josh Loomis of the National
Jewish Health Cytometry Core and Randi Anselment and Dr. Maki Nakayama for the TCR
sequencing. This work was supported in part by NIH grants AI-18785 (PM) AI092108 (LG) and
AI103736 (LG).
Author Contributions
JSB provided the idea for these experiments and made the constructs to produce the transgenic
mice, LG provided the idea for these experiments, helped in analyses and helped write the
manuscript, JWK wrote software to analyze the TCR sequences, helped in analyses and
helped write the paper, JW sorted T cells, tested the role of TCR in MHC restriction and
performed the PCR reactions to analyze TCR sequences, EK analyzed the mice, JC, SL and
TD helped analyze the data, PM designed the experiments, analyzed the data and wrote the
manuscript.
A.
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A
8204
*
MHC TCR
BL/6
b
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DC
Number of mature cells per thymus (x 10-6)Number of mature cells in lymph nodes
(x 10-6)
20 4020 40
MHC
TCR
b
f
s
CD4 CD8
*
**
*
CD4 CD8MHC TCR
b
f
s
***
***
*** ***
**
*
10 20
10 20 10 20 30 CD4/CD8 in thymus or lymph nodes
b
f
s
***
***
***
Thymus Lymph nodes
***
302010 1 2 3
Figure 1. CD4 selection in mice expressing single TCRchains and
different MHC alleles. Cells were isolated from the thymuses and lymph nodes
of mice expressing a single TCR, DOWT or DO48A , and different MHC
haplotypes and stained for expression of CD4 and CD8 and CD44. Results are
the means and standard errors of the mean (SEMs) of three independently
analyzed mice expressing the indicated TCRs and MHC alleles. Student t
analyses were used to compare results between the DOWT and DO48A
paired samples. *p<0.05, **p<0.01, ***p<0.001.
% of CD4 or CD8 T cells in lymph nodes
that are CD44hi
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
MHC
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
TCR
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MHCII
challenge None b f s
Host
MHCII
20
10
b
20
10
4
8
f
s
** **
**
*
(% of DOWT transduced - % of DO48A transduced)
DO48A CD4 T cells from hosts of the indicated MHC
haplotype that respond to spleen cells expressing the
indicated MHC haplotype.
Figure 2. TCRcontributes to the MHCII allele bias of selected
naïve CD4 T cells. Naïve CD4 T cells were isolated from the lymph
nodes of DO48A H2 b, f or s mice and incubated for 2 days in
wells coated with anti-TCRand anti-CD28. Thus activated, the T
cells were spinfected with GFP-expressing retroviruses expressing
also DOWTor DO48A. The cells were cultured for a further 2
days and then challenged with spleen cells from mice expressing
the indicated MHC alleles, or in the absence of added spleen cells.
One day later the cells were stained for expression of CD69.
Results were calculated as the (% of GFP+ T cells transduced with
DOWT-expressing retroviruses that were CD69+) (the % of
GFP+ T cells transduced with DO48A-expressing retroviruses that
were CD69+) in wells containing the same challenge spleen cells.
Shown are the average results +/- standard error of the mean
(SEM) from 3 independent experiments. *p<0.05, **p<0.01 by one
way ANOVA followed by Newman Keuls analyses.
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(>150,000)
1000
Total Sequences analyzed (x10-3)
50
60
40
20
Accumulated Unique
Sequences (x10-3)
B6 (H-2b)
A
Accumulated Unique
Sequences (x10-3)
4
8
12
Total Sequences analyzed (x10-3)
100050 100050 100050
H-2bH-2fH-2s
B C D
48A (~4,600)
WT (~30,000)
48A (~5,600)
WT (~17,400)
48A (~6,200)
WT (~6,800)
Figure 3. Expression of a single TCR chain, DOWT and, even more
markedly, DO48A, reduces the number of different TCRchains that can be
positively selected, regardless of the selecting MHCII allele. Naïve CD4 T
cells were isolated from the spleens (B6) or lymph nodes of mice expressing MHC
b, f or s, single TCR chains and heterozygous for expression of functional TCR
chains. Their expressed TCR chains were sequenced and analyzed with
species accumulation curves. Results were combined from 3 independently
sequenced data sets from mice of each genotype except for those for H2s
DOWT animals, which were combined from only two independently sequenced
animals. Data are shown together with an estimate (bracketed) of the total
numbers of different TCR protein sequences present in the naïve CD4 T cells of
each type of mouse.
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A
Jaccard similarity coefficient
0.1 0.2 0.3 0.1 0.2 0.3
b
f
s
b
f
s
b
f
s
DOWT DO48A
TCR
MHCII of:
Comparator Comparatee
b
f
s
***
*** ***
***
***
***
***
***
***
***
0.4 0.8
0.4 0.8
Anne Chao Jaccard abundance based index
B
b
f
s
b
f
s
b
f
s
DOWT DO48A
TCR
MHCII of:
Comparator Comparatee
b
f
s
*** ***
*** ***
***
***
***
***
***
***
***
TCRs selected on:
IAb IAf IAs
0.1 0.2 0.3
0.4 0.8
Comparator Comparatee
DOWT DOWT
DO48A ***
DO48A DOWT
DO48A **
***
Jaccard similarity coefficient
***
IAb IAf IAs
Comparator Comparatee
DOWT DOWT
DO48A ***
DO48A DOWT
DO48A ******
TCRs selected on:
TCRPartner in selection
TCRPartner in selection
Anne Chao Jaccard abundance based Index
C
D
***
0.4 0.8 0.4 0.8
0.1 0.2 0.3 0.1 0.2 0.3
Fig. 4. TCR sequences on naïve CD4 T cells are determined by the
selecting MHCII allele and the co-selected TCR. TCRs on naïve CD4 T
cells from the lymph nodes of TCR+/- mice expressing a single TCR and
various MHC alleles were sequenced and analyzed as described in Fig. 3.
Results are the means and SEMs of three independently sequenced animals of
each genotype except for H2s DOWT animals, of which only two mice were
analyzed. ***p<0.001 by one way ANOVA with Newman-Keuls post analysis.
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Fig. 5 The frequency with which TRAVs are used on naïve CD4 T cells in TCRtransgenic
mice depends on their selecting MHCII and their partner TCR.TCRson preselection
thymocytes or naïve CD4 T cells from the lymph nodes of TCR+/- mice expressing a single
TCRand various MHC alleles were sequenced and analyzed as described in Fig. 3. A. Shown
are the Euclidean distances for TRAV use between the data for individual mice. Samples are
hierachically ordered. Individual mice of the same genotype are numbered m1-3. B. The
average % use of each TRAV in mice expressing the indicated MHCII allele and TCR. Results
are the means +/- SEMs of 3 identical mice, except for H2s DOWT animals, for which results
are the averages of 2 mice. TRAVs are ordered by family, not by position on the chromosome.
B6
sDO48A
bDOWT
b DO48A
f DOWT
f DOWT m1
f DOWT m2
fDOWT m3
s DOWT m1
s DOWT m2
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m1
s DOb48A m1
bDOWT m1
b DOWT m2
b DOWT m3
B6
sDO48A
bDOWT
b DO48A
f DOWT
f DOWT m1
f DOWT m2
fDOWT m3
s DOWT m1
s DOWT m2
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m1
s DOb48A m1
bDOWT m1
b DOWT m2
b DOWT m3
Preselection
thymocytes
Naïve
CD4
LN T
cells
Preselection
thymocytes Naïve CD4 LN T cells
30
20
10
0
Euclidean
Distance
1
10
% of sample
fDO48A
TRAVs arranged by family
121 121
Color
TRAV
Family
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
21
C
D E
F G
b DO48AB
100
1
10
100
1
10
100
A
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b DO48A
B6
b DOWT
sDO48A
b DO48A
s DOWT m1
s DOWT m2
f DOWT
f DOWT m1
f DOWT m2
fDOWT m3
b DOWT m1
b DOWT m2
b DOWT m3
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m2
s DOb48A m3
Preselection
thymocytes
Naïve
CD4 LN T
cells
30
20
b DO48A
B6
b DOWT
sDO48A
b DO48A
s DOWT m1
s DOWT m2
f DOWT
f DOWT m1
f DOWT m2
fDOWT m3
b DOWT m1
b DOWT m2
b DOWT m3
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m2
s DOb48A m3
10
0
Euclidean
Distance
A.
Fig. 6 Naïve CD4 T cells in DOWT or DO48A mice preferentially use TRAJs
from the distal end of the TRAJ locus. TCRs on preselection thymocytes or naïve
CD4 T cells from the lymph nodes of TCR+/- mice expressing a single TCRand
various MHC alleles were sequenced and analyzed as described in Fig. 3. A. Shown
are the Euclidean distances for TRAJ use between the data for individual mice.
Samples are hierachically ordered. Individual mice of the same genotype are
numbered m1-3. B-L. The % use of each TRAJ in mice expressing the indicated
MHCII allele and TCR. Results are the means and SEMs of 3 identical mice, except
for H2s DOWT animals, for which results are the averages of 2 identical mice.
TRAJs are ordered by position on the chromosome. Also shown are the means and
SEMs of TRAJ use by 5 independently sequenced preselection thymocytes and 3
independently sequenced naïve CD4 T spleen T cells from B6 mice.
6
6
40
30
10
40
B. Preselection thymocytes
C. CD4 Naïve spleen cells
G. Naïve CD4 H2bDO48A
H. Naïve CD4 H2fDO48A
60
E. Naïve CD4 H2fDOWT
4
4
2
20
20
20
30
10
20
10
40
20
10
58 2
TRAJ
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0.4 0.80.4 0.8
B
b
f
s
b
f
s
b
f
s
DOWT DO48A
MHCII of:
Comparator Comparatee
b
f
s
*** ***
*** ***
***
***
***
***
***
***
Comparison of CDR3sequences on T
cells expressing:
Anne Chao abundance based index
A
Jaccard similarity coefficient
0.1 0.2 0.3 0.1 0.2 0.3
b
f
s
b
f
s
b
f
s
DOWT DO48A
MHCII of:
Comparator Comparatee
b
f
s
***
*** ***
***
***
***
***
***
***
***
Comparison of CDR3sequences on T
cells expressing:
***
IAb IAf IAs
0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2
Comparator Comparatee
DOWT ***
**
***
Jaccard similarity coefficient
TCRco-selected partner
C
DOWT
DO48A
DOWT
DO48A
DO48A ***
Comparison of CDR3sequences on T cells
bearing DOWT versus DO48A selected on:
***
IAb IAf IAs
Comparator Comparatee
***
***
***
TCRco-selected partner
Anne Chao Jaccard abundance based index
D
0.4 0.8 0.4 0.8 0.4 0.8
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
Comparison of CDR3sequences on T cells
bearing DOWT versus DO48A selected on:
***
Fig. 7. CDR3 sequences on naïve CD4 T cells are determined by the selecting MHCII
allele and the co-selected TCR. TCRs on naïve T cells from mice expressing a single
TCR and various MHCII alleles were sequenced and analyzed for their CDR3sequences
as described in Fig. 3 and 4. CDR3 sequences were defined as the amino acids between
and including the conserved cysteine at the C terminal end of the TRAV and the conserved
phenyl alanine, tryptophan or leucine in the TRAJ region. Shown are the means and SEMs of
3 independently sequenced identical mice except for H2s DOWT mice, in which case only 2
mice were analyzed. ***p<0.001, **p<0.01 by one way ANOVA with Newman-Keuls post
analysis.
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B
Jaccard similarity
coefficient
0.2 0.2 1.0
b
f
s
b
f
s
b
f
s
MHCII of:
Comparator Comparatee
b
f
s
***
*** ***
***
***
*** ***
***
0.60.4 Anne Chao Jaccard
abundance based
index
0.4 0.8
0.2 0.4
A
b
f
s
b
f
s
b
f
s
MHCII of:
Comparator Comparatee
b
f
s
*** ***
*** ***
***
***
***
***
Anne Chao Jaccard
abundance
based index
Jaccard similarity
coefficient
DOWT T cells expressing
TRAV6-7DN TRAJ12 DOWT T cells expressing
TRAV7-2D TRAJ13
0.4 0.8
0.2 0.3
C
b
s
b
s
MHCII of:
Comparator Comparatee
b
s
***
Anne Chao Jaccard
abundance
based index
Jaccard similarity
coefficient
DO48A T cells expressing
TRAV6-7DN TRAJ12
***
0.1
***
***
0.4 0.80.2 0.4
E
TCRpartner of:
Comparator Comparatee
DOWT ***
Anne Chao Jaccard
abundance
based index
Jaccard similarity
coefficient
T cells selected on IAb and
expressing TRAV14-3A TRAJ22
*
***
*
DOWT
DO48A
DOWT
DO48A
DO48A
0.4 0.80.1 0.2
FTCRpartner of:
Comparator Comparatee
DOWT ***
Anne Chao Jaccard
abundance
based index
Jaccard similarity
coefficient
T cells selected on IAf and
expressing TRAV14-3A TRAJ22
**
***
**
DOWT
DO48A
DOWT
DO48A
DO48A
0.3
*
0.4 0.8
0.2 0.4
D
b
s
b
s
MHCII of:
Comparator Comparatee
b
s
***
Anne Chao Jaccard
abundance
based index
Jaccard similarity
coefficient
DO48A T cells expressing
TRAV7-2D TRAJ11
-
***
***
***
Fig. 8. N region amino acids in CDR3of naïve CD4 T cells are determined by
the selecting MHCII allele and the co-selected TCR. TCRs on naïve CD4 T
cells of mice expressing a single TCR and various MHCII alleles were sequenced
as described in Figs 3 and 4. Mice were as listed in those Figs. Comparisons were
made of N regions derived from the same TRAV TRAJ pair providing that all mice in
the comparisons expressed at least 5 different sequences involving the chosen
TRAV TRAJ pair. Results shown are the means +/- SEMs of the data from identical
mice. Statistical analyses involved one way ANOVA tests with Newman-Keuls post
test analyses (A, B) or Student t tests (C-F). *p<0.05, **p<0.01, *** p<0.001.
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% of unique sequences in: B6 B6.AKR B6.NOD
B6 (IAb) 35.9+/-4.6 18.9+/-2.9 10.3+/-1.6
B6.AKR (IAk IEk) 35.8+/-2.8 12.2
B6.NOD (IAg7) 36.6+/-2.8 23.1
% of total sequences in: B6 B6.AKR B6.NOD
B6 (IAb) 62.1+/-2.9 44.1+/-2.5 26.7+/-1.6
B6.AKR (IAk IEk) 57.8+/-2.1 27.0
B6.NOD (IAg7) 54.9+/-2.1 41.2
That are also found in:
That are also found in:
Supplementary Table 1
In normal mice, a significant number of TCR sequences appear on naïve CD4 T
cells regardless of the selecting MHCII allele
Naïve CD4 T cells were isolated from the lymph nodes of normal mice of the
indicated strains and their TCR sequences identified as described in the
Methods section. Shown are the %s of unique sequences and the %s of total
sequences that were shared between pairs of mice of the indicated strains. Data
were obtained from 3 independently sequenced B6 mice and one each B6.AKR
and B6.NOD animals and are the means and standard errors of the means of the
comparisons.
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TRBV (Arden nomenclature) CDR3 TRBJ
DOWT 13.2 (V8.2) CASGSGTTNTEVFF 1.1
DO48A 13.2 (V8.2) with Y48 mutated to A CASGSGTTNTEVFF 1.1
Supplementary Table 2
Sequences of TCR transgenes
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025 50
Total Sequences analyzed (x10-3)
DOWT
IAb
IAf
IAs
2
4
6
8
10
2
4
6
8
10
2
4
6
8
10
Host
MHC
Accumulated unique sequences (x 10-3)
DO48A
DO48A
DO48A
DOWT
DOWT
Supplementary Fig. 1 The naïve CD4 T cells in mice
expressing a single TCRchain express a limited
number of TCRsequences regardless of the MHC
allele involved in their selection in the thymus.
Results were calculated as described in Fig. 3. Data
shown are for individual mice.
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Same MHC, Different TCR
Different MHC, DOWT
Different MHC, DO48A ***
***
***
***
Anne Chao Jaccard
abundance based index
Jaccard similarity
coefficient
0.02
0.01 0.04
0.02 0.06
Supplementary Fig. 2 TCRsequences are somewhat
more likely to be shared between T cells selected on the
same MHCII allele but differing in TCRthan between T
cells sharing TCRbut selected on different MHCII alleles.
TCRsequences were obtained and analyzed as described in
Figure 4. Data shown are combined as indicated between all
the mice illustrated in Fig.4 Results are the means and SEMs
of the data. ***p<0.001 by one way ANOVA with Newman Keuls
post analysis.
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b B6
b DO48A
1
10
% of sample
TRAVs arranged by family
121
1
10
1
10
1
10
1
10
Color
TRAV
Family
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
19
21
Supplementary Fig 3. Different TRAVs are
detected at different frequencies in preselection
thymocytes. Preselection DP thymocytes were
obtained from individual mice expressing the
indicated MHCII alleles and TCRs. Shown are the
%s with which individual TRAVs were detected.
Subfamily members of each family are color coded
as shown. Note, the TRAVs are ordered by family
and not by their position in the TCRlocus.
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H2f DO48A m1
H2f DO48A m2
H2f DO48A m3
H2b DO48A m1
H2b DO48A m2
H2b DO48A m3
H2b DOWT m1
H2b DOWT m2
H2b DOWT m3
H2s DO48A m1
H2s DO48A m2
H2s DO48A m3
H2f DOWT m1
H2f DOWT m2
H2f DOWT m3
H2s DOWT m1
H2s DOWT m2
H2f DO48A m1
H2f DO48A m2
H2f DO48A m3
H2b DO48A m1
H2b DO48A m2
H2b DO48A m3
H2b DOWT m1
H2b DOWT m2
H2b DOWT m3
H2s DO48A m1
H2s DO48A m2
H2s DO48A m3
H2f DOWT m1
H2f DOWT m2
H2f DOWT m3
H2s DOWT m1
H2s DOWT m2
Supplementary Fig. 4. TRAV usage by naive CD4 T cells
depends on the selecting MHCII allele and partner
TCR. The %s with which individual TRAVs were used by
mature naïve CD4 T cells in mice expressing different
MHCII alleles were plotted against each other for individual
mouse pairs. Individual mice of each genotype were
numbered m1-m3, numbers for each mouse are used
consistently throughout.
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DOWT
MHC
b f s
1 2 3 1 2 3 1 2
DO48A
MHC
b f s
1 2 3 1 2 3 1 2 3
MHC b
WT
1 2 3 1 2 3
DOTCR
48A
MHC f
WT
1 2 3 1 2 3
DOTCR
48A
MHC s
WT
1 2 3 1 2
DOTCR
48A
Supplementary Fig. 5 TRAVs are used to different extents by naïve CD4 T cells in mice expressing different MHCII alleles
and/or different TCRs. Data were obtained and analyzed as described in Figs. 3 and 5. DESeq 2 was used to compare the
frequencies with which different TRAVs were used by naïve CD4 T cells in mice expressing different MHCII alleles and/or different
TCRs. Differential expression analyses were performed using the DESeq2 package (v1.8.1) in the R language (v3.2.2). DESeq2
fit negative binomial regression models to each feature to compare between groups. Corrections for size factors for each sample
(animal) to account for differences in repertoire size were applied. The negative binomial dispersion parameter for each feature
was then calculated, sharing information across features with similar expression levels to moderate extreme empirical dispersion
estimates. Wald tests were applied to each feature to test for differential expression between groups. Features were considered
differentially expressed if they had a Benjamini-Hochberg adjusted p-value (i.e., false discovery rate) <0.05.
A. B. C. D. E.
5 25 50 75 95
% of normalized counts
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Supplementary Fig. 6. TRAJ usage by naive CD4 T cells
depends on the selecting MHCII allele and partner
TCR. The %s with which individual TRAJs were used by
mature naïve CD4 T cells in mice expressing different
MHCII alleles were plotted against each other for individual
mouse pairs.
b DOWT m1
b DOWT m2
b DOWT m3
f DOWT m1
f DOWT m2
fDOWT m3
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m2
s DOb48A m3
s DOWT m1
s DOWT m2
b DOWT m1
b DOWT m2
b DOWT m3
f DOWT m1
f DOWT m2
fDOWT m3
f DO48A m1
f DO48A m2
f DO48A m3
b DO48A m1
b DO48A m2
b DO48A m3
s DOb48A m1
s DOb48A m2
s DOb48A m3
s DOWT m1
s DOWT m2
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DOWT
MHC
b f s
1 2 3 1 2 3 1 2
DO48A
MHC
b f s
1 2 3 1 2 3 1 2 3
MHC b
WT
1 2 3 1 2 3
DOTCR
48A
MHC f
WT
1 2 3 1 2 3
DOTCR
48A
MHC s
WT
1 2 3 1 2
DOTCR
48A
DOWT
MHC
b f s
1 2 3 1 2 3 1 2
DO48A
MHC
b f s
1 2 3 1 2 3 1 2 3
A. B. C. D. E.
5 25 50 75 95
% of normalized counts
Supplementary Fig. 7 TRAJs are used to different extents by naïve CD4 T cells in mice expressing different MHCII
alleles and/or different TCRs. Data were obtained and analyzed as described in Figs. 3 and 5. DESeq 2 was used as
described in supplementary Fig. 5 to compare the frequencies with which different TRAJs were used by naïve CD4 T cells in
mice expressing different MHCII alleles and/or different TCRs.
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certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under
The copyright holder for this preprint (which was notthis version posted August 15, 2017. ; https://doi.org/10.1101/176388doi: bioRxiv preprint
***
***
**
***
***
***
***
***
***
**
***
***
***
*
***
**
***
***
***
*
***
**
***
***
***
***
***
**
***
***
***
51510
Average # amino acids in
CDR3(C to F inclusive)
Average # N region
bases in CDR3
123
TCRMHCII Organ
B6 b
DOWT b
f
s
b
f
s
DO48A
TCRMHCII Organ
B6 b
DOWT b
f
s
b
f
s
DO48A
Thymus
Spleen
Thymus
Spleen
LN
LN
LN
LN
Supplementary Fig 8. CDR3length on naïve CD4 T cells
depends upon the selecting MHC allele and the co-selected
TCR. Data were obtained as described in Figs. 3 and 4.
Shown are the average lengths of CDR3and N region bases
on naïve CD4 T cells coexpressed with the indicated TCR.
Results are the means and SEMs of 3 mice of the same
haplotype except for s DOWT mice, in which case the results
from 2 mice were averaged. Errors are calculated with one way
ANOVA with the Bonferroni post test comparing all pairs. * =
p<0.05, **=p<0.01, ***=p<0.001. Statistical results are omitted
for pairs which were not significantly different from each other.
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Supplementary Fig. 9. N region amino acids in CDR3of naïve CD4 T cells are
determined by the selecting MHCII allele and the co-selected TCR. TCRson
naïve CD4 T cells of mice expressing a single TCRand various MHCII alleles were
sequenced as described in Figs 3 and 4. Mice were as listed in those Figs. The
results for samples expressing the same TRAV TRAJ pairs for all comparisons in
which each mouse expressed at least 5 different sequences involving the chosen
TRAV/TRAJ pair were averaged. Results shown are the means +/- SEMs of the
data from identical mice. Statistical analyses for comparisons involving 3 different
types of mice involved one way ANOVA tests with Newman-Keuls post test analyses.
Student t tests were used to analyze statistically the differences between pairs of
mice. ** p<0.01, *** p<0.001.
A
Jaccard similarity
coefficient
0.1 0.2 1.0
b
f
s
MHCII of:
Comparator Comparatee
b
f
s
***
*** ***
***
***
*** ***
***
0.6
Comparison of TCRN region
sequences on DOWT T cells
expressing the same TRAV and TRAJ
0.2 Anne Chao Jaccard
abundance based index
0.3
***
*** ***
***
0.1 0.2 1.0
b
f
s
b
f
s
b
f
s
MHCII of:
Comparator Comparatee
b
f
s
***
*** ***
***
*** ***
0.6
Comparison of TCRN region
sequences on DO48A T cells
expressing the same TRAV and TRAJ
0.2
***
na na
na
na
***
IAb IAf IAs
0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.2
Comparator Comparatee
DOWT ***
***
***
Jaccard similarity coefficient
TCRco-selected partner
C
DO48A **
Comparison of TCRN region sequences on T
cells expressing DOWT versus DO48A and
the same TRAVs and TRAJs
0.3
***
***
IAb IAf IAs
Comparator Comparatee
***
**
***
Anne Chao Jaccard Abundance based Index
D
0.4 0.8 0.4 0.8 0.4 0.8
**
DOWT
DO48A
b
f
s
b
f
s
Jaccard similarity
coefficient Anne Chao Jaccard
abundance based index
DOWT
DO48A
Comparison of TCRN region sequences on T
cells expressing DOWT versus DO48A and
the same TRAVs and TRAJs
DOWT
DO48A
DOWT
DO48A
DOWT
DO48A
TCRco-selected partner
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Data 1
1
2
3
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6
7
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MHCII b b f s b f s
TCRB6 DOWT DO48A
4
5
6
% of TCRs with indicated amino acid
100
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100
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50
CDR3
Position
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b b f s b f s
B6 DOWT DO48A
CDR3
Position
4
5
6
7
100
50
100
50
100
50
100
50
Frequency of amino acid use in CDR3s with lengths (between the C terminal TRAV C
and the TRAJ conserved F/W/L inclusive) of:
Supplementary Fig. 10 The frequency of amino acid use in CDR3on naïve CD4 T cells depends on the
selecting MHCII allele, the co-selected TCRand the length of the CDR3.Data from TCRsequences
obtained as described in Fig. 4 were used to analyze the frequency with which different amino acids were used at
different positions.
Data 1
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T
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R
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P
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A
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b b f s b f s
B6 DOWT DO48A
4
5
6
7
8
CDR3
Position
100
50
100
50
100
50
100
50
100
50
13 amino acid CDR314 amino acid CDR312 amino acid CDR3
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