Multiple Glycines in TCR ?-Chains Determine Clonally
Diverse Nature of Human T Cell Memory to Influenza A
Yuri N. Naumov,2* Elena N. Naumova,‡Maryam B. Yassai,†Kalyani Kota,*
Raymond M. Welsh,* and Liisa K. Selin*
Detailed assessment of how the structural properties of T cell receptors affect clonal repertoires of Ag-specific cells is a prerequisite
for a better understanding of human antiviral immunity. Herein we examine the ? TCR repertoires of CD8 T cells reactive against
the influenza A viral epitope M158–66, restricted by HLA-A2.1. Using molecular cloning, we systematically studied the impact of
?-chain usage in the formation of T cell memory and revealed that M158–66-specific, clonally diverse VB19 T cells express ?-chains
encoded by multiple AV genes with different CDR3 sizes. A unique feature of these ? TCRs was the presence of CDR3 fitting to
an AGA(Gn)GG-like amino acid motif. This pattern was consistent over time and among different individuals. Further molecular
assessment of human CD4?CD8?and CD4?CD8?thymocytes led to the conclusion that the poly-Gly/Ala runs in CDR3? were
a property of immune, but not naive, repertoires and could be attributed to influenza exposure. Repertoires of T cell memory are
discussed in the context of clonal diversity, where poly-Gly/Ala runs in the CDR3 of ?- and ?-chains might provide high levels
of TCR flexibility during Ag recognition while gene-encoded CDR1 and CDR2 contribute to the fine specificity of the TCR-peptide
MHC interaction. The Journal of Immunology, 2008, 181: 7407–7419.
clusters between T cells and APCs (1–4). The T cells that have an
identical clonal origin express a unique ?? TCR that defines clonal
fine specificity to Ag. Multiple clones with diverse, and to some
extent overlapping, specificities provide protective immunity
against viral infections. After viral clearance, a number of epitope-
specific clones are retained, thus creating long-lasting memory
TCR repertoires. It is currently agreed that clonal survival during
and after viral clearance is a final result of multiple factors. Among
these factors is the molecular nature of ?? TCRs.
Ag-driven clonally expressed ? TCR repertoires have been in-
tensively examined in experimental animal models (5–7) and hu-
man diseases (8–11). These studies have concluded that repetitive
antigenic challenges correlate with increased frequencies of Ag-
specific, clonally diverse cells that share amino acid sequences
D8 T cells express ?? TCRs that bind to immunogenic
peptides loaded into class I MHC molecules (pMHC)3
and initiate formation of the supramolecular activation
within CDR3 of their expressed ? TCRs “fitting best” to epitope
recognition. Since these memory cells are at high precursor fre-
quencies and usually have lower TCR-mediated activation require-
ments than do naive cells, they provide rapid pathogen clearance in
the case of reinfection. Although ? TCR-mediated selections in
response to the immunogenic epitopes are well documented, little
is known about ? TCR involvement in selection of human CD8 T
Crystallization of TCR-pMHC has revealed that ?-chains might
provide a significant contribution to the interactive interface, vary-
ing from 37% to 74% of the total surface (12–16). This implies that
? TCR usage might be a critical element that defines whether
Ag-reactive cells are saved in a memory compartment. In this
study, we sought to investigate ? TCRs expressed by memory
cells, and we observed several previously unknown properties that
might determine the clonal nature of memory repertoires.
Human CD8 T cell reactivity against the influenza A matrix
(M1) protein-derived epitope, M158–66, represents an excep-
tional system to understand the molecular properties of ? TCRs
expressed by memory cells selected in humans. Because the M1
protein is highly conserved among influenza A viral strains,
reinfections during a lifetime (17) result in formation of the
strong CTL recall responses against the M158–66epitope prac-
tically in all HLA-A2 (HLA-A*0201) individuals (10, 11, 18–
20). For instance, by age 15 years HLA-A2 children possess a
well-established M1-specific memory pool comprised of CD8 T
cells expressing BV19 gene-encoded ?-chains (formerly BV17)
Our previous studies revealed that multiple VB19 clones spe-
cific to M158–66coexist in middle-aged individuals (22–24).
Those clones were defined based on the uniqueness of the nucle-
otide composition in the V-NDN-J regions encoding ?-chains.
Therefore, they were referred to as VB19 clonotypes since the ?
TCR usage remained unknown. These influenza-specific clono-
types utilize BV19 gene-encoded ?-chains with two CDR3 sizes
*Department of Pathology, University of Massachusetts Medical School, Worcester,
MA 01655;†Blood Research Institute, The Blood Center of Wisconsin, Milwaukee,
WI 53201; and‡Department of Public Health and Family Medicine, Tufts University
School of Medicine, Boston, MA 02111
Received for publication May 23, 2008. Accepted for publication September 15,
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants U19-AI057319 (to
Y.N.N.), U19-AI062627 (to Y.N.N. and E.N.N.) and AI45751 (to L.K.S.). The con-
tents of this publication are solely the responsibility of the authors and do not rep-
resent the official view of the National Institutes of Health.
2Address correspondence and reprint requests to Dr. Yuri N. Naumov, Department of
Pathology, University of Massachusetts Medical School, 55 Lake Avenue, Worcester,
MA 01655. E-mail address: Yuri.Naumov@umassmed.edu
3Abbreviations used in this paper: pMHC, immunogenic peptides loaded into class I
MHC molecules; 4SP, CD4?CD8?; 8SP, CD4?CD8?.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
fitting into IRSS- and IGS-like motifs. Although individual VB19
CD8 T clonotypes expressed structurally identical ? TCRs, they
have different M158–66:HLA-A2.1 tetramer (M1 tetramer) binding
capacity and peptide concentration-dependent proliferation in cell
cultures (23). This suggests that even if VB19 cells were selected
due to the best CDR3? “fit” to M158–66:HLA-A2 recognition,
their ? TCR usage could be different. Therefore, we reasoned that
memory cells from a single family, VB19, could be used to ex-
amine the breadth of the memory ? TCR repertoire and provide
insight on why ?-VB19 chains alone are not exclusive determi-
nants in clonal selection (10, 23).
It has been reported that the optimal ?? TCR interaction with
pMHC requires that CDR3? and CDR3? should have similar sizes
for proper engagement of V? and V? domains (25). Our present
study demonstrates that VB19 cells express AJ42 gene-encoded ?
TCRs, which contain multiple (up to five) poly-Gly/Ala runs in the
long CDR3?, allowing engagement of cells from different VA
families, thus utilizing different CDR1? and CDR2? in epitope
recognition. This observation occurred in five individuals whose
M1-specific cells were selected using M1 tetramer and VB19
mAbs. During the study period, we did not find preselection for
poly-Gly/Ala runs in the CDR3? in human CD4?CD8?thymo-
cytes from the T cell subset (VA27-JA42) that was most prominent
in response to flu-M158–66epitope. Taken together, our study led
to the conclusion that the selection of T cells possessing poly-Gly/
Ala runs within their CDR3 was driven in response to the influenza
A M158–66epitope rather than by biased gene recombination or
thymic selection. We propose that the presence of poly-Gly/Ala
runs in the CDR3s of ? and ? TCRs contributes to conformational
flexibility of Ag-specific receptors. This implies that a robust im-
mune response associates not only with T cells whose CDR3? and
CDR3?, which are generated during random gene recommenda-
tions, have the “fittest” complementarity to pMHC, but also with T
cells whose Ag receptors utilize germline gene-encoded regions if
CDR3s have high levels of flexibility.
Materials and Methods
Five healthy blood donors, donors A, B, C, D, and E (50, 47, 40, 56, and 26
years old, respectively), were defined to be HLA-A2.1 (HLA-A*0201)-posi-
tive based on MHC class I typing with the Biotest SSP (sequence-specific
primer) system (Biotest Diagnostics).
Thymic tissue was collected as a discard during reconstructive surgical
procedure on an HLA-A2.1-positive 3-mo-old child having a congenital
cardiac defect, under a protocol approved by the Internal Review Board of
The Children’s Hospital of Wisconsin.
Influenza A peptide M158–66-specific CD8 T cell cultures
CD8 T cells were isolated from peripheral blood collected from donors
A–E using anti-CD8 microbeads (Miltenyi Biotec), following the manu-
facturer’s recommendations. The purity of isolated CD8 T cells usually
was ?95%. To generate peptide-specific cell lines, CD8 T cells (0.25 ?
106cells/ml) were cocultured with TAP1/TAP2-defective target T2 (174 ?
CEM.T2, American Type Culture Collection (ATCC)) cells (0.05 ? 106
cells/ml) in 4 ml complete culture medium/well. The T2 cells were incu-
bated overnight with M158–66peptide (1 ? 10?6M). Before the setting of
M1-specific cultures, peptide-pulsed T2 cells were irradiated (3000 rad)
and intensively washed to avoid residual peptide. The complete culture
AIM-V medium (Invitrogen) contained human rIL-2 (10 U/ml) (BD
Pharmingen) and was supplemented with 14% supernatant from the IL-2-
producing MLA 144 cell line (TIB 201, ATCC). The CD8 T cell lines were
cultured in 12-well plates and split with rIL-2-containing media every 3
days. Once a week cultures were restimulated with peptide-coated, irradi-
ated T2 cells. The cultures where T2 cells were not coated with peptide
served as controls.
The peptide M158–66(GILGFVFTL) was synthesized on Pepsyn KA resin
(BioSource International) using a 9050 PepSynthesizer (Millipore). Pep-
tide was purified by reverse-phase HPLC (?90% purity) using a C18 col-
VB19 mAb and M158–66/HLA-A2 tetramer staining
To examine the frequency of M158–66-specific VB19 cells, CD8 T cells
were sampled from the cultured lines and costained with M158–66/HLA-
A2.1 tetramer (M1 tetramer) (Beckman Coulter) and VB19 family-specific
mAbs (Immunotech) according to the manufacturers’ recommendations.
Initially cells were stained with allophycocyanin-labeled M1 tetramer for
20 min at room temperature, and then FITC-labeled VB19 mAbs were
added for an additional 20 min under the same conditions. The stained cells
were washed in a copious volume of FACS buffer (PBS, 2% FCS, 0.2%
sodium azide). Flow cytometry was performed using a FACSVantage and
FACSCalibur (BD Biosciences). To isolate M1-specific cells, CD8 T cell
line samples were stained with M1 tetramer and VB19 mAbs (donors A, B,
and C) or M1 tetramer alone (donor D), and then FACS sorted using a
MoFlo sorter (Dako). Purity of the FACS-sorted cells was ?95% (data not
Thymocyte staining and sorting
The thymus was disaggregated by passing through a wire mesh. Cells were
suspended in RPMI 1640 medium (Invitrogen), 0.1% sodium azide, and
2% FCS, and stained with mouse mAbs to human cell surface markers:
CD3-FITC conjugate, CD4-Tri-Color conjugate, and CD8-R-PE conjugate
(Caltag Laboratories). A three-color sort was performed using FACStar
(BD Biosciences), and single-positive CD4?CD8?and CD4?CD8?thy-
mocytes were collected. Primary gating was set on the CD3 marker, which
resolved the thymocytes into three populations, CD3?, CD3low, and
CD3high. The CD3highpopulation was further divided on the basis of CD4
and CD8 expression. Cells were collected into 0.5 ml of FCS so that the
final concentration in the tube was 10% (5 ml final volume).
RNA isolation, cDNA synthesis, and titration
CD8 T cells separated from peripheral blood, cultured lines, or FACS
sorting after M1 tetramer/VB19 mAbs staining were used for RNA isola-
tion and cDNA synthesis as previously described (22, 26). Genomic DNA
was prepared from FACS-sorted thymocytes treated with nucleic acid lysis
buffer (10 mM Tris (pH 8.2), 0.4 M NaCl, 2 mM EDTA) in the presence
of SDS and proteinase (26). Then the cells were incubated overnight at
45°C to ensure complete lysis. After the incubation, proteins were precip-
itated by adding 5.3 M NaCl, and DNA was precipitated from the super-
natant with ethanol (27). Before generation of ? TCR-specific CDR3 spec-
tratypes, genomic DNA and cDNA samples were titrated using
semiquantitative PCR amplification of the TCR CB region, as described
(26, 28). Briefly, serially diluted cDNA (or genomic DNA) aliquots were
amplified 24 cycles using forward and reverse CB-specific primers under
nonsaturating PCR conditions, and resolved on denaturing 50% urea/5%
polyacrylamide gels. The cDNA (or genomic DNA) aliquots of cDNA that
contained an equal quantity of total ? TCR transcripts were used for se-
quential CDR3? spectratype-generating PCR amplifications.
Detailed descriptions of CDR3 spectratyping conditions were published
previously (22, 26). Briefly, cDNA samples were amplified in a PCR cock-
tail that contained forward VA family-specific primer and reverse CA-
specific primer labeled from the 5? end with FAM. The nucleotide se-
quences of 34 VA family-specific and CA-specific primers were described
elsewhere (29). To examine the ? TCR repertoires of human thymocytes,
genomic DNA samples were amplified using VA27- and JA42-specific
primers. The PCR cycle consisted of 0.5 min at 94°C for denaturation, 0.5
min at 58°C for annealing, and 1.5 min at 72°C for elongation. The final
elongation was extended to an additional 7 min at 72°C. After 31 cycles of
PCR amplification, 10 ?l of VA-CA-amplified cDNA products were
loaded and run on 50% urea/5% polyacrylamide sequencing gels for 2 h 15
min to 3 h 45 min depending on VA-CA primer combination. This tech-
nique ensures CDR3? bands visualization after gel scanning on the fluo-
rescence detection system FluorImager 595 (Molecular Dynamics). Mul-
tiple CDR3? bands within a given VA family reflect clonal complexity
based on ? TCR transcript size heterogeneity.
PCR product cloning and DNA sequencing
cDNA samples generated from M1 tetramer/VB19 mAbs-stained, FACS-
sorted CD8 T cells (donors A–C), M1 tetramer-stained (donor D), and
7408TCR ?-CHAIN EXPRESSION IN MEMORY CD8 T CELLS
nonsorted cultured cells (donor E) were amplified with unlabeled CA-spe-
cific primer and one of VA family-specific primers in separate nonsaturated
PCR reactions and immediately subcloned into the plasmid vector pCR4-
TOPO (Invitrogen). The following cDNA libraries were generated and
screened: VA8 (includes VA8.1 and VA8.3), VA8.6, VA10, VA12 (in-
cludes VA12.1, VA12.2, and VA12.3), VA27, VA29, VA34, and VA35.
VB19-CDR3? plasmid subclones of donor A cultures were described else-
where (23). From 48 to 96 plasmid subclones from each VA cDNA library
have been sequenced using a Taq DyeDeoxy terminator cycle sequencing
kit (Applied Biosystems). Analysis of VA and JA regions flanking CDR3?
nucleotide sequences indicated ?0.25% divergence from the genomic se-
quences, which can be attributed to the MMLV reverse transcriptase and/or
TaqDNA polymerase infidelity. The assignment to AV and AJ gene fami-
lies of the subcloned and sequenced CDR3? plasmid inserts is given ac-
cording to ImMunoGeneTics (IMGT). CDR3? size count includes C (from
CASS) and F (FGXG) according to IMGT.
The VA and JA family origins of the influenza A M158–66-specific VB19
clonotypes from donors A–E are shown in supplemental Table I.4Here we
depict each clonotype number within VA cDNA libraries, their CDR3?
sizes (aa), amino acid (highlighted in red), and nucleotide sequences within
the AV-JA gene junction. The data sets corresponding to VA27-JA42 rep-
ertoires of the CD4?CD8?and CD4?CD8?thymocytes and CD8 T cells
from M158–66-specific cell culture (donor A, year 2004, 5 wk culture) are
given in supplemental Table II. The CDR3? loops are shown in red; non-
template-encoded amino acids in CDR3? are revealed in blue and are
underlined. Clonotype unique identifiers are labeled as ID.
? TCRs expressed by M158–66-specific VB19 cells originate
from multiple VA families
To define the breadth of the ? TCR repertoire of flu-specific cells
that express VB19 ?-chains with high affinity to the M158–66
epitope (19), we used CDR3? spectratyping. Lack of available VA
family-specific mAbs and the relatively low precursor frequency
of M158–66-specific cells varying in a range of 0.1–0.8% in the
peripheral blood CD8 T cells (30) were the two major reasons for
using cell lines. To generate a sufficient number of cells to screen
? TCR families responding to the flu epitope, the CD8 T cells
collected from five middle-aged HLA-A2 individuals were stim-
ulated with irradiated T2 cells charged with the M158–66peptide to
induce T cell division in vitro. After 3–5 wk in culture, cells were
either FACS sorted after costaining with M1 tetramer and VB19
mAbs (donors A–C), M1 tetramer alone (donor D), or used di-
rectly (donor E). In our experiments, the purity of FACS-sorted
cells gated as M1 tetramer?or M1 tetramer?VB19?populations
usually exceeded 95% (data not shown).
We reasoned that by using RT-PCR amplification of cDNA
samples with AV gene-specific primers, we would define ? TCR
transcriptional profiles of the cells proliferating upon peptide stim-
ulation and binding M1 tetramer. Therefore, cDNA samples gen-
erated from nonstimulated CD8 T cells and FACS sorted with M1
tetramer alone and with M1 tetramer/VB19 mAbs combined were
amplified with VA-specific primers for 29 of 52 human ? TCR
genes (29). To examine the ? TCR repertoires of the peripheral
4The online version of this article contains supplemental material.
specific CD8 T cells based on CDR3? size
heterogeneity (donor A). A, VA repertoire of
CD8 T cells isolated from peripheral blood
from donor A (year 2002). B and C, VA rep-
ertoire of the CD8 T cells FACS sorted from
influenza M158–66-specific cultures from do-
nor A (year 2002) using (B) M1 tetramer
alone (week 4) and (C) M1 tetramer and
VB19 mAbs combined (week 5). The num-
bers on top of the panels indicate the AV
gene origin of RT-PCR-amplified ? TCR
transcripts (per IMGT). Solid brackets indi-
cate dominant CDR3? bands that have been
subcloned and sequenced.
7409 The Journal of Immunology
blood, CD8 T cells, and cells FACS sorted from the epitope-spe-
cific cultures, we ran off the RT-PCR products on the CDR3?
spectratyping gels having assumed that the M158–66-specific cells
would be selected within VA families. The increased fraction of
M158–66-specific cells within each VA family was ascertained by
increased densities of the selected CDR3? sizes detected on the gel
images. To provide representative examples of the ? TCR reper-
toires of the unstimulated CD8 T cells and cells from the M158–66-
specific cultures, we present CDR3? spectratypes from donor A
(Fig. 1), whose ? TCR repertoire of peripheral blood CD8 T cells
is shown in Fig. 1A. The ? TCR transcriptional profiles of donor
A M1 tetramer?(FACS sorted, week 4 culture) and M1
tetramer?VB19?(FACS sorted, week 5 culture) cells across and
within VA families are shown in Fig. 1, B and C, respectively. The
numbers on the x-axis indicate the VA family origin of ? TCR
transcripts, while DNA bands resolved on the y-axis and their in-
tensities represent proportions of ? TCR transcripts with identical
CDR3 sizes within the respective families. Expecting that a rep-
ertoire of the peripheral blood CD8 T cells is extremely diverse,
we observed multiple CDR3? bands within each of the VA fam-
ilies examined. While only 56% of the VA repertoire (29 of 52
families) was examined, we observed that CD8 T cells were de-
rived from different VA families and utilized different CDR3?
sizes. Representation of each VA family was skewed, with a high
yield of VA24-, VA30-, and VA12-specific transcripts and a low
yield of the VA14-, VA16-, and VA40-specific transcripts (Fig.
1A). Interestingly, CDR3? spectratypes of the freshly isolated cells
from donors B–E have similar patterns to donor A, namely: com-
plexity of VA families, multiple CDR3? size usage within each of
the VA families, and variation of these values among individuals
(data not shown).
To gain insight about the M158–66-specific ? TCR repertoire,
we ran off RT-PCR products from M1 tetramer?CD8 T cells
(FACS sorted, week 4 culture, donor A) as shown in Fig. 1B. The
M1 tetramer?cells expressed ? TCRs encoded by multiple VA
families (VA8, 10, 14, 19–24, 12, 27, 8.6, 22, 38.2, 41, and 29).
Remarkably, M158–66-specific cells from a few families utilized ?
TCRs with different CDR3? sizes, as shown on VA12, VA27,
VA8.6, and VA29 spectratypes (Fig. 1B, brackets). To link the
mentioned ?-VA transcripts to VB19?cells, we sorted cells
costained with M1 tetramer and VB19 mAbs. A representative
example of ? TCR repertoire of the double-positive cells from
donor A is shown in Fig. 1C. Again, we observed that M158–66-
specific VB19?cells coexpressed ? TCRs encoded by different
VA families with different CDR3? sizes (Fig. 1C). Interestingly,
CDR3? spectratypes of M1 tetramer?cells (Fig. 1B) were similar
to those generated from the M1 tetramer?VB19?subpopulation
(Fig. 1C). We showed that RT-PCR products from M1 tetramer?
and M1 tetramer?VB19?cells were derived from VA8, VA10,
VA12, VA27, VA8.6, VA22, and VA29 families. Although we did
not align CDR3? bands within each VA spectratype, this obser-
vation reflects a complexity of the M158–66-specific recall ? TCR
repertoire with engagement of different VA domains in pMHC
This observation is evidence that flu-specific VB19?cells uti-
lized ? TCRs from different families, and the diverse CDR3? sizes
further confirm our previous observations that VB19?cells have
different clonal origins and avidities to the M158–66epitope (22–
24). Although we did not show ? TCR repertoires from donors
B–E whose CD8 T cells were isolated from the peripheral blood
and cell cultures, there were multiple ? TCR transcripts from
No peptide culture (wk 4)
M1 culture (wk 4)
No peptide culture (wk 4)
M1 culture (wk 3) M1 culture (wk 9) M1-tet, M1 culture (wk 5)
M1 culture (wk 3) M1-tet, M1 culture (wk 5)M1-tet/VB19 mAbs, M1 culture (wk 5)
VB19 mAbs, M1 culture (wk 5)
PCR negative control
2000 2001 2002
cells in M1-specific CD8 T cell cultures (donor A). A, VA27 spectratypes.
B, VA8.6 spectratypes. CD8 T cell cultures from donor A were generated
every 12 mo and tested for M158–66-specific reactivity based on CDR3?
spectratyping. The top numbers indicate the years when cultured lines were
generated. The spectratypes are given for the CD8 T cell cultures (M1
culture) and cultures where cells were FACS sorted using M1 tetramer
(M1-tet), VB19 mAbs, or both (M1-tet/VB19 mAbs). Sorted CD8 T cells
isolated from the peripheral blood (ex vivo) and cultured without peptide
(No peptide culture) were used as controls. The arrows indicate the sub-
cloned and sequenced CDR3? bands.
Time-independent immunodominance of VA27 and VA8.6
CDR3? sizes consistently respond against influenza M158–66:HLA-A2
epitope. A, VA27 spectratypes. B, VA8.6 spectratypes. CD8 T cell cultures
from donors B, D, and E were generated and tested for M158–66-specific
reactivity using CDR3? spectratyping. M1 culture indicates nonsorted
M158–66-specific CD8 T cell cultures; M1-tet corresponds to M1 tetramer?
FACS-sorted cultured cells. Sorted CD8 T cells from the peripheral blood
(ex vivo) and cultured without peptide (No peptide culture) were used as
the negative control. CDR3? bands from the marked areas (dotted boxes)
were subcloned and sequenced.
CD8 T cells from VA27 and VA8 families and different
7410 TCR ?-CHAIN EXPRESSION IN MEMORY CD8 T CELLS
v8.3 v12.2 v29
CDR3 size v27
v3v8.1 v12.1v12.3 v8.4
53% 38%13% 57%57% 40% 100% 25%
% of JA42+ clonotypes
0 1-50 51-99 100
domains. The JA42?clonotype distributions based on CDR3? sizes and responding VA families are shown for each individual (A) and overall (B). The labels
in the top row indicate VA family according to IMGT. The total numbers of clonotypes and JA42?clonotypes for each VA family are shown in the rows with
gray background. The total numbers of all (VA), JA42?(JA42) clonotypes, and their contribution (%) are shown in the three last columns. The CDR3? sizes (aa)
expressed by flu-M158–66-reactive VB19 clonotypes are shown in the first column. B, Illustrates the ? TCR repertoire summary in a similar manner. In both A
and B, each cell represents the number of clonotypes by VA family and CDR3 size and is color-coded. The color-coding system (C) represents the number of
clonotypes classified into five categories (1, 2–4, 5–8, 9–11, and 12? clonotypes) shown in rows and the proportions of corresponding JA42?clonotypes also
classified into four categories (0, 1–50, 51–99, 100%) shown in columns in three degrees of shading.
The distribution of ? TCR repertoires of the influenza-specific VB19 CD8 T cells from the HLA-A2?individuals as a function of ?-chain V
7411The Journal of Immunology
Table I. Poly-Gly/Ala runs in the entire CDR3? expressed by influenza M158–66-specific VB19?CD8 T cellsa
aM158–66-specific clonotypes, from five HLA-A2 individuals, derived from different VA families share Gly/Ala amino acid runs in CDR3? of different sizes. Amino acid
residues in CDR3? loops are shown in red. The Ala and Gly in CDR3? are underlined. Clonotype unique identifiers are labeled as ID. The total numbers of M158–66-specific
VB19?clonotypes within correspondent VA-cDNA libraries and their CDR3? nucleotide and deduced amino acid sequences are also available as supplemental Table I.
7412TCR ?-CHAIN EXPRESSION IN MEMORY CD8 T CELLS
M158–66-specific cells regardless of whether they were M1 tet-
ramer/VB19-positive mAbs. Of note, since HLA-A2.1 T2 cells
were used as APCs in our cell cultures, their ? TCR transcripts
could be misinterpreted as specific to M158–66reactivity and affect
the general picture of the recall ? TCR repertoires. Therefore, we
screened ? TCR transcripts from T2 cells and defined that neither
of the VA-specific primers used amplified ? TCRs. We concluded
that ? TCRs from 4- to 5-wk cultures were derived from M158–66-
specific CD8 T cells.
Since allelic exclusion is not applicable to ? TCR gene rear-
rangement, the peripheral T cells express two ?-chain mRNAs
(derived from both chromosomes), and 25–30% of cells express
two ? TCR proteins paired with a single ? TCR where only one
?? TCR heterodimer binds self-MHC (31–33). Considering the
previous report where M1-specific VB19 clones were found to
express primarily ?-VA27 (formerly VA10.2) (11), it was some-
what surprising that VA27 ? TCR mRNA/cDNA transcripts did
not dominate within the ? TCR transcriptional pools (Fig. 1, B and
C). We reasoned that if the recall response was mediated by nu-
merous VB19?clonotypes, then detection of multiple VA tran-
scripts would be expected. However, if clonal cells were selected
due to the molecular nature of their ? TCRs, then similarity in ?
TCR repertoires would be revealed even if they were collected
from different individuals. Thus, we tested ? TCR repertoires from
four HLA-A2 blood donors. We thought that if only the VA27
family was involved in M158–66recognition, then the probability
that flu-specific cells from different donors would have identical
second ? TCRs (same VA families and CDR3? sizes) would be
extremely low. However, if cells were selected based on ? TCR
fitness to flu-M158–66:HLA-A2, then they would have similar
CDR3? sizes and amino acid sequences of ? TCRs.
First, we reexamined whether flu-M1-reactive cells consistently
utilized CDR3? of different sizes. Peptide-specific cultures from
donor A were regenerated 12 mo apart and reexamined regarding
? TCR usage. The representative CDR3? spectratypes of VA27
and VA8.6 cells isolated from blood, mock, and M158–66-specific
cultures are shown in Fig. 2. We found that immunodominant
CDR3? bands had identical sizes in all CD8 T cell lines and prop-
erly aligned with those from M1 tetramer?sorted cells (Fig. 2,
arrows). Based on spectratyping patterns, we concluded that
VA27?cells evenly utilize three different CDR3? sizes, while
VA8.6 cells express mostly long CDR3?.
To further verify that flu-M1-reactive cells derived from differ-
ent VA families express CDR3? of different sizes, we generated
and screened VA spectratypes of the cultured cells from donors
B–E. The representative VA27 and VA8.6 spectratypes of M1
tetramer?VB19?(donor B), M1 tetramer?(donor D), and non-
sorted cultured (donor E) CD8 T cells, including controls, are
shown in Fig. 3. After M1 tetramer?cells were sorted from cul-
tures from donors B and C, we intentionally gated on M1
tetramerhighVB19highT cell populations. Again, we observed that
flu-specific cells expressed AV27, 10, 8.6, 12, and 29 gene-encoded
? TCR transcripts (not shown).
From these findings, we concluded that frequencies of VA27
and other families were low in peripheral blood and control cul-
tures, since we used equalized quantities of RNA/cDNA tran-
scripts from mock and peptide-specific lines. However, stimulation
with influenza-derived M158–66peptide rapidly induced peptide-
dependent proliferation of T cells derived from diverse VA fam-
ilies. Although VA27 spectratypes with three CDR3? bands were
remarkably similar between donors, we observed that patterns of
VA8.6 and other VA usage varied between individuals.
Table II. The nontemplate-encoded Gly/Ala runs in the CDR3?
expressed by influenza M158–66-specific CD8 T cellsa
IDsize (aa) amino acid sequence
aInfluenza-specific M1 tetramer?VB19?CD8 T cells express ?-chains from 11
VA families with size-restricted CDR3?. Amino acid residues in CDR3? loops are
shown in red and blue. Nontemplate-encoded Ala and Gly are shown in blue and are
interlined. CD8 T cells were isolated from M158–66-specific culture using M1 tet-
ramer and VB19 mAbs (donor A).
7413The Journal of Immunology
Influenza M158–66-specific ? TCR repertoire: preferential
selection for CDR3? size and poly-Gly/Ala runs
To better understand how influenza-specific cells might utilize
?-chains encoded by different AV genes and having different
CDR3? sizes, we used cDNA samples from M1 tetramer?VB19?,
M1 tetramer?, and bulk M158–66-specific cultured CD8 T cells to
generate VA family-specific cDNA libraries and sequenced CDR3
inserted into plasmid vector. Note that our methodology does not
allow assigning TCR ?- and ?-chains to a single clone; however,
we were confident that utilized cells were influenza M158–66-spe-
cific given that CDR3? spectratypings were overrepresented by
VB19 family (Ref. 23 and data not shown). Hereafter, CDR3?
sequences are referred to as “clonotypes” based on the uniqueness
of nucleotide composition in the AV-N-AJ gene recombination
sites. We thought that the number of CDR3? sequences within the
VA cDNA library might serve as a proxy of the clonotypes’ rel-
ative frequencies within the respective VA family, if the ? TCR
repertoires possess a polyclonal nature. This also could validate
M158–66-driven clonotype selection mediated through CDR3?
amino acid compositions. The complete data sets of VA and JA
usage, CDR3? sizes, and amino acid sequences of ? TCRs ex-
pressed by M158–66-reactive clonotypes from all five donors are
available in supplemental Table I.
We have demonstrated that the ? TCR repertoire reactive
against influenza was complex and included multiple VB19?
clonotypes from multiple VA families with different CDR3? sizes.
Hereafter, we examined repertoire structure where clonotypes
from the JA42 family were considered as de facto M158–66-spe-
cific. Thus, we tabulated the number of clonotypes that share
CDR3? sizes within VA families and determined proportions of
the JA42?clonotypes across VA families and across CDR3?
sizes. In Fig. 4 we illustrate the clonotype distribution by VA
families and by CDR3? sizes (from 18 to 10 aa) for each donor
(Fig. 4A) and overall (Fig. 4B) in a color-coded manner (Fig.
4C). The color-coding system represents the number of clono-
types classified into five categories (shown in rows) and the
proportions of corresponding JA42?clonotypes also classified
into four categories (shown in columns in three degrees of shad-
ing). We also provide the summaries for the total number of all
VA clonotypes, JA42?clonotypes, and their contribution (%)
in all VA cDNA libraries by each CDR3? size and overall for
each donor (Fig. 4A) and for all five donors (Fig. 4B). For
example, donor A had 105 clonotypes, out of which 67 (64%)
clonotypes were JA42?; the dominant CDR3? size was 15 aa,
found in 40 clonotypes, of which 33 (83%) were JA42?(shown
We observed that the total number of detected clonotypes varied
from 5 to 105, and JA42?clonotypes represented from 23% to
64% of all ? TCR repertoires. Among all donors, the distribution
of clonotypes reflects that M158–66-specific T cells express
CDR3? of different sizes; however, CDR3? with 12, 14, and 15 aa
were dominant (Fig. 4A). The dominance of specific VA families
and specific CDR3? sizes was well pronounced in the overall sum-
mary (Fig. 4B). We found that all VA8.1?clonotypes, 67% of
VA27?clonotypes, and VA12.3?clonotypes expressed JA42-en-
coded ? TCRs. The JA42 usage associated with CDR3? sizes of
12 aa (80%), 15 aa (59%), and 14 and 16 aa (36% and 33%,
The high utilization of JA42?clonotypes with different CDR3?
sizes from different VA families (on average 53%) upon epitope
stimulation suggested that expression of VA27 ?-chains by
VB19?CD8 T cells is not an exclusive requirement to M158–66-
specific reactivity. We also determined the following hierarchy of
CDR3? sizes as a function of VA family origin, such as 15 ? 12 ?
14 aa residues. Moreover, if the VB19?clonotypes from the JA42
family are considered as M158–66-specific, then the hierarchy of
VA usage can be presented as the following rule: VA27 (three
CDR3? sizes and JA42) ? VA8.6 and VA35 (two CDR3? sizes
and JA42) ? VA8.1 to VA29 (only CDR3? of 15 aa and JA42).
To investigate whether proper combination of CDR3? sizes and
amino acid sequences is a strict requirement in M158–66recogni-
tion, while CDR1? and CDR2? usage is less stringent, we aligned
?-chains of JA42?clonotypes originating from different VA fam-
ilies from all five donors (Tables I) involved in this study and from
donor A (Table II) alone. Strikingly, many clonotypes contained
poly-Gly/Ala runs where only two Gly were AJ42-gene encoded
specific clonotypes expressing long CDR3? chains (14 and 15 aa)
contained nontemplate-encoded Gly/Ala runs (Table II, underlined
characters) with other nonpolar, polar, and charged amino acids.
Importantly, although ? TCRs might have identical amino acid
sequences in CDR3, they were encoded by different nucleotide
sequences generated during AV-N-AJ42 gene recombination that
serves as clonotype marker (available as supplemental Table I).
Table III. Alignment of 11 VA chains involved in M158–66:HLA-A2 recognitiona
SS SVFSS LQWYRQEPGEGPVLLVT VVTGGE VKKLKRLTFQFGDARKDSSLHITAAQ PGDTGLYLC AG
SS SIFNT WLWYKQEPGEGPVLLIA LYKAGE LTSNGRLTAQFGITRKDSFLNISASI PSDVGIYFC AGQ
YS SSVSVY LFWYVQYPNQGLQLLLK YLSGSTL VESINGFEAEFNKSQTSFHLRKPSVH ISDTAEYFC AVS
YS YGGTVN LFWYVQYPGQHLQLLLK YFSGDPL VKGIKGFEAEFIKSKFSFNLRKPSVQ WSDTAEYFC AVN
YS YGATPY LFWYVQSPGQGLQLLLK YFSGDTL VQGIKGFEAEFKRSQSSFNLRKPSVH WSDAAEYFC AVG
YT VSPFSN LRWYKQDTGRGPVSLTI MTFSEN TKSNGRYTATLDADTKQSSLHITASQ LSDSASYIC VVS
YS NSASQS FFWYRQDCRKEPKLLMS VYSSG NEDGRFTAQLNRASQYISLLIRDSK LSDSATYLC VVN
YS DRGSQS FFWYRQYSGKSPELIMF IYSNG DKEDGRFTAQLNKASQYVSLLIRDSQ PSDSATYLC AVN
YS NSAFQY FMWYRQYSRKGPELLMY TYSSG NKEDGRFTAQVDKSSKYISLFIRDSQ PSDSATYLC AMS
YT NSMFDY FLWYKKYPAEGPTFLIS ISSIKD KNEDGRFTVFLNKSAKHLSLHIVPSQ PGDSAVYFC AAS
SS KTLYG LYWYKQKYGEGLIFLMM LQKGGE EKSHEKITAKLDEKKQQSSLHITASQ PSHAGIYLC GAD
CDR1α loop CDR2α loop CDR3α loop
aM158–66epitope-specific CD8 T cells from eleven VA families express structurally different CDR1? and CDR2? based on amino acid composition. CDR1?, CDR2? and
CDR3? (amino termini) are shown in bold.
7414TCR ?-CHAIN EXPRESSION IN MEMORY CD8 T CELLS
Based on the collected and analyzed data sets, we defined the
following rules in CDR3? usage: AGAGGGG in CDR3? with 15
aa; AGAGGG in CDR3? with 14 aa; and AGGG in CDR3? with
12-aa residues. Although we generated and screened the VA
cDNA libraries of different sizes for each individual (Fig. 4A), this
pattern was consistent between the studied subjects (Table I).
Thymic VA27-JA42 repertoire assessment: poly-Gly/Ala run in
CDR3? is a property of M158–66:HLA-A2 epitope-selected
The presence of nontemplate poly-Gly/Ala runs in TCR ?-chains
expressed by flu-specific T cells could either reflect the CDR3?
amino acid sequence distribution in naive T cells emerging from
the thymus, or it could reflect a preferential selection of these
sequences during flu-specific immune responses. To examine
whether poly-Gly/Ala runs are a property of M158–66-specific
memory, we examined AV27-N-AJ42 gene recombination in hu-
man CD4?CD8?and CD4?CD8?thymocytes. We reasoned that
if poly-Gly/Ala is an intrinsic property of the long CDR3?, then
this motif could be defined in CD4?CD8?(4SP) and CD4?CD8?
(8SP) thymocytes. Therefore, thymic tissue was used for 4SP and
8SP thymocyte isolation, genomic DNA preparation, and PCR am-
plification, using VA27 and JA42 family-specific primers. The
flow cytometry data and corresponding VA27-JA42 spectratypes
are shown in Fig. 5. In addition to thymic spectratypes, we used
spectratypes of the M158–66-specific culture and sorted M1
tetarmer?VB19?T cells that served as the positive controls for
As shown in Fig. 5B, the proportions of 4SP and 8SP thymo-
cytes that have CDR3? composed of 12-aa residues were under-
represented within the VA27?JA42?population, yet they were
abundant in bulk epitope-specific culture and within M1
tetramer?VB19?populations. To further examine the presence of
poly-Gly/Ala runs in conjunction with CDR3? sizes, we cloned
and sequenced 400 and 300 CDR3? plasmid subclones from the
8SP and 4SP thymocytes, respectively. Here, we defined 129 “in-
frame” rearrangements for 8SP thymocytes and 89 “in-frame” re-
arrangements for 4SP thymocytes. This frequency was expected
since two of three rearrangements generate nonproductive CDR3?.
As a control, we also used VA27-JA42 subclones from bulk M158–66-
specific culture (donor A, year 2004, 5-wk culture) and M1
tetramer?VB19?sorted populations (donor A, year 2002, 4-wk
culture). The ? TCR repertoire profiles of the VA27-JA42?clono-
types from the 8SP and 4SP thymocytes and cultured CD8 T cells
are available as supplemental Table II.
Although 8SP thymocytes utilizing CDR3? of 14- and 15-aa
residues were dominant based on spectratyping results (Fig. 5B),
more clonotype diversity was observed among cells using CDR3?
neity of the VA27-JA42 CD4?CD8?
and CD4?CD8?thymocytes. A, Flow
cytometry data of the FACS-sorted
cytes. B, VA24-JA42 spectratyping of
bulk CD8 T cells from peptide-spe-
cific culture, M1 tetramer?VB19?
population from the cells line. 8SP
cytes, respectively. Left-side arrows
and numbers indicate ? TCR tran-
scripts that encode 15, 14, and 12 aa
in CDR3?. ctrl indicates PCR nega-
tive control. The CDR3? sizes are
shown from C (CASS) to first F
CDR3? size heteroge-
7415 The Journal of Immunology
of 12-aa residues (20%, 13 of 66 clonotypes) and 15-aa residues
(31%, 21 of 66 clonotypes) (supplemental Table II, CD4?CD8?
thymocytes). The similar weak association between CDR3? band
intensity on spectratyping and clonotype diversity was seen for
4SP thymocytes (supplemental Table II, CD4?8?thymocytes).
Only one dominant clonotype detected among 8SP thymocytes
represented 11% (14 of 129 sequences) of the population and used
short CDR3? (11 aa) created by bland AV27 and AJ42 gene
Since we were interested to examine whether TCR interaction
with HLA-A2 would preferentially select cells based on CDR3?
sizes with nontemplate-encoded Gly/Ala runs, we examined the
occurrence of poly-Gly/Ala runs by plotting frequencies of these
amino acids, for all clonotypes, as a function of the CDR3? sizes
(Fig. 6). The Gly/Ala runs could be encoded if linking the AV27
gene (CAG) with the AJ42 gene segment (GGSQG. . . ) occurs
after deletion of one nucleotide from the 3? end of the AV27 gene
and seven nucleotides from the 5? end of AJ42 gene. Therefore, we
expected to observe Gly and Ala strings in a short CDR3?. How-
ever, we were interested whether the long (14–15-aa) CDR3?
were enriched by nontemplate Gly/Ala. As shown in Fig. 6A, the
frequency of nontemplate Gly/Ala in 8SP thymocytes with 14–15
aa in CDR3? was below detection level, considering the number of
identified clonotypes, similar to what was observed for 4SP (0–
5%) clonotypes (Fig. 6B). However, proportions of cells with non-
template Gly and Ala were in the range of 40–60% (12 of 17
clonotypes) for M1 tetramer?VB19? cells (Fig. 6C) and 40–90%
(10 of 26 clonotypes) for CD8 T cells that proliferated in the pep-
tide-specific culture (Fig. 6D). Therefore, we conclude that the
increased frequency of Gly/Ala runs is associated with flu
M158–66-driven selection rather than with V-J recombination and
A detailed assessment of the TCR repertoires of Ag-specific T
cells is a prerequisite for a better understanding of human an-
tiviral immunity. Here we systematically examined the ? TCR
repertoires of memory CD8 T cells reactive against the influ-
enza A viral epitope, M158–66, restricted by HLA-A2.1. The
M158–66-specific, clonally diverse VB19 CD8 T cells expressed
?-chains from several VA families with different CDR3 sizes. A
unique feature of these ? TCRs was the presence of poly-Gly/
Ala runs in the CDR3, fitting to an AGA(Gn)GG-like amino
acid motif. These nontemplate-encoded poly-Gly/Ala runs in
the CDR3 of the M158–66-specific memory pool were signifi-
cantly enriched over those in naive thymocytes, indicating that
Gly/Ala runs provided a selective advantage in Ag-driven rep-
ertoire development in the periphery. These poly-Gly/Ala runs
in the CDR3 of ?- and ?-chains might provide enhanced TCR
flexibility during Ag recognition.
The mechanisms that shape T cell memory through ? TCR se-
lection have been difficult to delineate due to the technical re-
straints associated with the lack of VA-family specific mAbs and
T cell ability to coexpress two ?-chains (31, 33, 34). Nevertheless,
our molecular cloning techniques demonstrate that the influenza A
M158–66-specific T cell memory contains a number of additional
features contributed by ? TCR diversity. These TCR ?-chains that
paired with the VB19 ?-chains were of 11 VA families with three
remarkably different sizes in CDR3? (Fig. 4B). Given that the
M1-specific clonotypes from different VA families express differ-
ent CDR1? and CDR2? (Table II), proper accommodation of dif-
ferent CDR1? and CDR2? to the M158–66-:HLA-A2 might occur
if the CDR3? could undergo conformational adjustment. In this
regard, enrichment of Gly and Ala might provide increased struc-
tural flexibility in CDR3? and satisfy this criterion.
It is commonly accepted that the fine specificity of epitope rec-
ognition is due to structural complementarity of CDR3? and
CDR3? to MHC-presented immunogenic peptides under condi-
tions where CDR1 and CDR2 orient the TCR ?- and ?-chains to
MHC molecules. In contrast to Abs that usually have large sur-
faces with complementarity to their cognate Ags (35), only 21–
34% of the ?? TCR’s surfaces are in direct contact with pMHC
complexes (16, 36). Moreover, the contributions of CDR3? and
CDR3? are relatively small, representing 21% and 24% on
average of V? and V? domains, respectively. These properties of
VA27-JA42 thymocytes. A, CD4?CD8?thymocytes, (B) CD4?CD8?thy-
mocytes, (C) CD8 T cells from M158–66-specific culture (donor A, year
2004, week 3), (D) M1 tetramer?VB19?CD8 T cells (donor A, year 2002,
M158–66-specific culture, week 5). The values in the lower x-axis indicate
the CDR3? sizes (aa). “All” corresponds to the total number of clonotypes
defined within VA27-JA42 cDNA libraries. The numbers on the Y-axis
indicate occurrences of only Gly, only Ala, Gly and Ala, and neither Gly
nor Ala as a percentage of all nontemplate-encoded amino acid residues in
CDR3? for clonotypes with identical CDR3? sizes. The total numbers of
the VA27-JA42 clonotypes sharing CDR3a sizes are shown on the top of
each bar. The data reflecting Gly, Ala, and Gly/Ala in the CDR3 have been
extracted from the data sets available as the supplemental material (sup-
plemental Table II (for A–C) and supplemental Table I (donor A) (for D)).
Frequencies of Gly, Ala, and Gly/Ala in CDR3? of the
7416 TCR ?-CHAIN EXPRESSION IN MEMORY CD8 T CELLS
TCR-pMHC interactions impose strict requirements on ?- and
?-chains. For example, side chains of amino acids located with-
in the CDR3 must have optimized sizes and charges to interact with
the foreign peptide, and CDR3 of ?- and ?-chains ought to have
the similar sizes (25). However, we show examples where short
CDR3? (12 aa) pair with one amino acid longer VB19-CDR3?
(counting from C (CAS) to F (FGXG)), similar to crystallized ??
TCR expressed by M158–66-specific clone JM22 (37). The CD8 T
cells from VA27 and VA8 families in five studied individuals
mostly expressed these short CDR3a loops. In contrast, longer
CDR3? sequences (14 and 15 aa) were found with VA families
other than VA27 and VA8. These “non-VA27” VB19?T cells
share ?-JA42 chains with VA27 T cells (Fig. 4B), but express
different CDR1 and CDR2 of their ?-chains (Table III). Given that
VB19 cells express rigid CDR3? fitting into the “IRSS-” or
“IGS”-like motifs, a plausible explanation that structurally differ-
ent V? domains are used to recognize influenza epitope is that the
CDR3? loops undergo significant conformation (38) associated
with poly-Gly/Ala runs. We therefore suggest that the CDR3?
bearing long poly-Gly/Ala strings allow CDR1? and CDR2? en-
coded by different VA families to be used during influenza Ag
recognition, and we discuss the theoretical basis for this suggestion
AJ42 gene-encoded products are not unique in the sense of con-
taining two Gly runs, since 7 out of 51 AJ genes encode two, and
even three, Gly. However, 59% (37 of 63) of the clonotypes with
CDR3? with 15 aa belong to this particular JA42 family (Fig. 4B).
It seems that the combination of long CDR3? with poly-Gly/Ala
runs provides flexibility for ?? TCR to bind to the M158–66:
HLA-A2 epitope, since we defined only eight clonotypes from
“non-VA27” families (namely, VA10, 8.6, VA34 and VA35) that
expressed short CDR3? of 12 aa (Fig. 4B). Gly is a unique amino
acid because it lacks a side chain, and Ala contains only a methyl
group as a side chain. It has been shown that proteins whose func-
tions depend on adjustment to ligands often contain flexible loops.
Usually Gly is located within these loops, providing flexibility in
protein-protein or protein-ligand interactions. For example, poly-
Gly strings have been found in the HIV protease flap region, in
?1,4-galactosyltransferase-I, fructose-1,6-bisphosphate aldolase,
and other enzymes (39–41). TCR contact with the pMHC mole-
cule also follows the same rule. Thermodynamic studies of three
TCR-pMHC binding, including ?? TCR from M158–66-specific
clone JM22, revealed that this process correlates with considerable
conformational adjustment in CDR3? and CDR3? (42, 43). For
instance, Reiser and coworkers reported that KB5-C20 (TCR spe-
cific to pKB1/H-2Kb) exhibits large conformational alteration in
the CDR3? (six amino acids longer than CDR3?) for proper ac-
commodation to pMHC (15). Recently, the same group reported
that similar structural flexibility might be observed in CDR3?
(BM3.3 specific to two peptides with low sequence similarity pre-
sented by H-2Kb) (13). Based on these studies, the authors con-
cluded that ?? TCR propensity to modify its complementarity sur-
face, mostly in the CDR3, might be the origin of ?? TCR intrinsic
ability to interact with the different epitopes. In line with these
studies is the observation of structural flexibility of the ?? TCRs
expressed by human T cell clones reactive against Tax11–19pep-
tide (from HTLV) presented by HLA-A2.1 (44, 45). Remarkably,
these clones expressed ? TCRs (VB13.1) that contained a PGxG
motif in the CDR3? and efficiently recognized the original epitope
and its variants. Another proof outlining the importance of TCR
structural flexibility in epitope recognition comes from the crys-
tallization of the ?? TCRS expressed by clone LC13 specific to
EBNA-3339–347peptide presented by HLA-B8. In this case, Ala-
GlyGly runs were contained in the CDR3? (46).
The occurrence of poly-Gly runs in ? TCRs with a long CDR3
is attributed to the D region where VDJ gene transcription in three
“open-reading-frames” would encode multiple Gly. This rule,
however, cannot be applied to ? TCRs lacking D-encoded regions.
The existence of VB19 clones specific to flu-M158–66that utilize
? TCRs from different VA families provides an interesting exam-
ple of epitope recognition where germline-encoded segments of
?-chains (i.e., SQG from AJA42 gene) contact M158–66, while
segments created by AV/AJ42 recombination are positioned out-
side M158–66:HLA-A2 and could be flexible due to Gly/Ala en-
richment (37, 42). A recent study of the ?? TCR (from clone JM22
specific to M158–66-epitope) before and after binding to M158–66:
HLA-A2 revealed that the CDR3? loop swiveled and made an ?5
Å outward shift (37, 42). Note that the CDR3? loop from JM22
contains only 12 aa. Therefore, we are confident that the CDR3?
with 14 and 15 aa defined in our study might have considerably
more rotation and movement during M158–66:HLA-A2 binding
and allow the CDR1? and CDR2? from different VA families to
interact with HLA-A2 ?1 helix.
A less explored field in human immunology is the analysis of
the molecular nature of the preimmune ? TCR repertoire. In our
study, we could not exclude the possibility that poly-Gly/Ala
runs might be a result of preferential AV27/AJ42 recombination
where long CDR3? might have increased frequencies of Gly
and Ala. If this were the case, we would have expected to see
increased observations of Gly and Ala in CD4?CD8?and
CD4?CD8?thymocytes regardless of class I and II HLA re-
strictions. Importantly, we examined the transcriptional profiles
of ? TCRs where VA27/JA42 transcripts might encode func-
tional (restricted by HLA-A2.1) and nonfunctional TCR
?-chains. Following extensive sequencing analysis of AV27/
AJ42 gene recombination and considering CDR3? sizes, we
concluded that Gly and Ala have similar frequencies with other
amino acids encoded by nontemplate segments of the VA27/
JA42 rearranged genes (Fig. 6 and supplemental Table II). Al-
though two Gly are derived from the AJ42 gene, as was ex-
nontemplate segments of long CDR3? (i.e., 14 and 15 aa) were
not Gly- and Ala-enriched. Therefore, we conclude that selec-
tion for poly-Gly/Ala runs was driven in response to M158–66
epitope during influenza exposure rather than by a gene
The flexibility of the ?? TCR structure might be an important
factor in the fate of memory T cells. In the case of influenza
M158–66-specific cells, the poly-Gly/Ala runs do not contact
M158–66or HLA-A2 directly, based on the crystallization of the
representative M158–66-specific JM22 clonal ?? TCR and its
variants (37, 42). Although the recognition M158–66:HLA-A2 is
a function of VB19 ?-chain (?70% of interactive interface), the
? TCRs with a highly flexible CDR3 might be used to recognize
structurally different Ags, thus contributing to the pattern of T
cell cross-reactivity, which we have observed in “heterologous
immunity” (47). If this is the case, influenza-specific VB19 T
cells with IRSS in CDR3? might engage CDR1? and CDR2?
during recognition of other p:HLA-A2, perhaps further enhanc-
ing the immunodominance of VB19 T cell clones.
Here we propose a three-step model explaining TCR interaction
with the M158–66/HLA-A2.1 complex (Fig. 7). In the first step,
CDR1? and CDR2? (?-VB19) contact the ?2 helix of HLA-A2.1,
pivoting the CDR3? to the peptide wherein R98 anchors the
?-chains to HLA-A2 and S99 interacts with the M158–66peptide.
In the second step, the SQG (AJ42 gene encoded) anchors the
CDR3? loop to Gly61 (M158–66), and the long CDR3? (poly-Gly/
Ala) undergoes conformational change. Since AGA(G/A)G in
VA27/JA42 transcripts, the
7417The Journal of Immunology
CDR3? imposes minimal energy requirement to change shape, this
leads to the third step, where engagement of different CDR1,2?
(from any of the VA domain) is sufficient for final TCR:M158–66/
HLA-A2.1 docking. The key element of this model is a long poly-
Gly/Ala moiety, which allows CDR3? to be extremely flexible
and adjust different TCR V? domains to the same pMHC com-
plex. If long CDR3? and/or CDR3? are more flexible and ac-
commodate ?? TCRs to different pMHC shapes and charges,
then cross-reactivity against different Ags might be a major
factor in memory formation. Of note, in our studies we also
defined, based on tetramer binding, “non-VB19” cells that were
able to recognize the M158–66/HLA-A2 epitope. Remarkably,
they also expressed long (15-aa) CDR3? with a GXGG motif
(Y. N. Naumov, unpublished).
The early studies of the cytotoxic CD8 T cell lines and clones
reactive against M157–68-expressing targets cells revealed that
M157–68peptide modifications in positions 58–60 were well tol-
erated, while modifications in positions 61–65 could abrogate
CTL response. In the last case, however, diminished cytotoxicity
was not absolute and depended on amino acid substitutions. Inter-
estingly, the T cell clones derived presumably from VB19 family
have different patterns of epitope dependency in the CTL assay
(10). Our own study demonstrated that small populations of the
VB19 T cells are able to proliferate and to produce IFN-? in re-
sponse to influenza M158–66and EBV-BMLF1280–288epitopes
(30). Although we did not yet define the structure of the cross-
reactive ?? TCRs, these observations suggest that conformational
flexibility of ?? TCRs and clonal diversity of reactive T cells
might be the best way to cope with different Ags.
In conclusion, we suggest that the immune response evolves in
a way where it engages T cells with structurally different ?? TCRs
specific to cognate Ags leading to an intrinsic capacity of these T
cells to interact with different pMHC shapes and charges. It is
tempting to speculate that the presence of multiple memory CD8 T
cell clones of diverse specificities due to adjustable Ag receptors is
the best way to optimize immune memory to ever-changing anti-
We thank Dr. Jack Gorski and Dr. Martin Hessner for scientific discussion,
and K. Bateman for editorial assistance with the manuscript.
The authors have no financial conflicts of interest.
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7419The Journal of Immunology