The Journal of Immunology
Diversity of the CD8+T Cell Repertoire Elicited against an
Immunodominant Epitope Does Not Depend on the Context
Brian D. Rudd,*,†,1Vanessa Venturi,‡,1Megan J. Smithey,* Sing Sing Way,x
Miles P. Davenport,‡and Janko Nikolich-Zˇugich*
The diversity of the pathogen-specific T cell repertoire is believed to be important in allowing recognition of different pathogen
epitopes and their variants and thereby reducing the opportunities for mutation-driven pathogen escape. However, the extent to
which the TCR repertoire can be manipulated by different vaccine strategies so as to obtain broad diversity and optimal protection
is incompletely understood. We have investigated the influence of the infectious/inflammatory context on the TCR diversity of the
CD8+T cell response specific for the immunodominant epitope in C57BL/6 mice, derived from glycoprotein B of HSV-1. To that
effect, we compared TCR V segment utilization, CDR3 length, and sequence diversity of the response to natural HSV-1 infection
with those elicited by either Listeria monocytogenes or vaccinia virus expressing the immunodominant epitope in C57BL/6 mice.
We demonstrate that although the type of infection in which the epitope was encountered can influence the magnitude of the CD8+
T cell responses, TCR b-chain repertoires did not significantly differ among the three infections. These results suggest that widely
different live vaccine vectors may have little impact upon the diversity of the induced CTL response, which has important
implications for the design of live CTL vaccine strategies against acute and chronic infections.
2010, 184: 2958–2965.
largely empirical. CD8+T cells play a central role in combating
intracellular infections and tumors, and much attention has been
devoted to understanding the immune correlates of protection and
efficacious CD8+T cell control of infection. Historically, CD8+
T cell responses have been evaluated largely on the basis of their
magnitude, with immune responses eliciting a greater number of
Ag-specific CD8+T cells generally considered more favorable.
T cell response does not always correlatewith immune defense (1),
The Journal of Immunology,
lthough vaccination provides the most successful means
for prevention of infectious disease, the development of
and this quantitative measurement by itself does not reflect the un-
now generally accepted that one key determinant of CD8+T cell-
mediated immune protection correlates with the diversity of TCR
use, because TCR diversity within an epitope-specific response
may enrich for higher avidity T cells (1), limit viral escape mutants
6). Collectively, these reports suggest that describing potential
clonotypes may help enable rational vaccine design.
A major challenge for vaccine development against chronic viral
infections like HIVand HCVis to induce responses characterized by
a diverse CD8+T cell repertoire, which is more likely to recognize
is especially difficult in those with physiological (e.g., the elderly,
because aging of the immune system is associated with decreased
TCR diversity and functionality) or pathophysiological/iatrogenic
(immunodeficiency, chemotherapy, bone marrow transplantation)
the diversity of the CD8+TCR repertoire can be manipulated by
different vaccine strategies has never been thoroughly investigated.
IFA) and dispersible (CpG, monophosphatidyl lipid A) adjuvants
preferentially skewed the TCR repertoire toward clonotypes that
exhibited higher TCR avidity. Importantly, these findings addressed
the effects on CD4+T cell repertoire of subunit vaccines and ad-
juvants. Viral “escape” from immune recognition is, however, most
prevalent in the context of CD8+T cell responses. Moreover, most
opposed to adjuvants. To address this question with regard to CD8+
*Department of Immunobiology and the Arizona Center on Aging, University of
Arizona College of Medicine, Tucson, AZ 85724;†BIO-5 Institute, University of
Arizona, Tucson, AZ 85719;‡Centre for Vascular Research, University of New South
Wales, Kensington, New South Wales, Australia; and
Center for Infectious Disease and Microbiology Translational Research, University
of Minnesota School of Medicine, Minneapolis, MN 55455
xDepartment of Pediatrics,
1B.D.R. and V.V. contributed equally to this work.
Received for publication October 27, 2009. Accepted for publication January 6,
This work was supported by United States Public Health Service Award AI066096
from the National Institute of Allergy and Infectious Diseases (to J.N-Zˇ.), the Aus-
tralian Research Council, and the Australian National Health and Medical Research
Council. J.N-Zˇ. is the Elizabeth Bowman Professor in Medical Sciences. M.P.D. is
a Sylvia and Charles Viertel Senior Medical Research Fellow. V.V. is an Australian
Research Council Future Fellow.
Address correspondence and reprint requests to Dr. Janko Nikolich-Zˇugich, Depart-
ment of Immunobiology, University of Arizona College of Medicine, 1656 East
Mabel Street, Tucson, AZ 85719. E-mail address: nikolich@E-mail.arizona.edu
Abbreviations used in this paper: gB, glycoprotein B of the herpes simplex virus; gB-
8p, immunodominant gB epitope, SSIEFARL; L. monocytogenes-gB, recombinant
Listeria monocytogenes carrying the gB-8p epitope; PCC, pigeon cytochrome c;
VACV-gB, recombinant vaccinia virus carrying the gB-8p epitope; WG, tryptophan-
T cells and live vaccines, we examined the extent of TCR diversity
elicited in response to different infectious vectors by sequencing
CD8+T cells specific for the HSV-1 immunodominant epitope de-
rived from glycoprotein B (gB498–505; referred to henceforth as the
gB-8p epitope) from mice infected with Listeria monocytogenes (L.
monocytogenes-gB) or vaccinia virus (VACV-gB) expressing the
immunodominant peptide (SSIEFARL). Both of these vectors have
previously been shown to be protective against a subsequent chal-
lenge with HSV (9, 10); however, the effects of these different in-
fections on the TCR repertoire have not been studied.
To better understand the molecular complexity of CD8+T cell
responses, our present analysis focused on two specific questions: 1)
Do infections with various recombinant vectors alter the diversity of
the epitope-specific CD8+TCR repertoire (i.e., do they modify the
number of clonotypes responding, or the clonal dominance structure
oftheresponse)?; and 2)Doesthe epitope-specific CD8+response to
different infections select different TCRs (i.e., do the responding
clones have different patterns of CDR3 amino acid sequences)?
an uninfected mouse (11, 12), of which 15–900 different TCRs re-
spond to a given epitope (12, 13). The link between the selective
forces involved in the preferential recruitment and expansion of dif-
ferent TCR clonotypes and their degree of avidity for cognate Ag
(14, 15) suggests likely variation among infections withdifferent Ag
of CD8+T cell division. We investigated whether these differences
viruses would likely favor the proliferation of CD8+T cells bearing
different TCR clonotypes.
Unexpectedly, we found comparable TCRb repertoire diversity
the response, and route of inoculation. Moreover, the characteristics
of the gB-8p–specific TCRb repertoires associated with HSV-1 in-
fection were shared by the gB-8p–specific TCRb repertoires from
mice infected with L. monocytogenes-gB and VACV-gB. These re-
sults have profound implications for the design of live vaccines
eliciting the most diverse CD8+T cell responses or covaccinating
with common escape variations may be a more efficacious strategy
for generating long-lived protective immunity against chronic viral
infections than altering the nature or type of vaccine vector.
Materials and Methods
Mice and infections
Male C57BL/6 (B6, H-2b) were purchased from the National Cancer In-
stitute (Frederick, MD) and maintained under pathogen-free conditions in
the animal facility at the University of Arizona (Tucson, AZ). Naive mice
were used at 8–10 wk of age, and experiments were conducted by
guidelines set by the University of Arizona Institutional Animal Care and
Use Committee. HSV-1 strain 17 was obtained from Dr. D.J. McGeoch
(University of Glasgow, Glasgow, U.K.), cloned as a syn+variant, and
titered on Vero cells in our laboratory, as previously described (16, 17).
Recombinant vaccinia virus expressing the MHC class I–restricted CTL
epitope HSV gB498–505 (SSIEFARL, gB-8p in this paper), designated
VACV-gB, was generously provided by Dr. S.S. Tevethia (Pennsylvania
State University College of Medicine, Hershey, PA). VACV-gB viral stocks
were propagated and quantified in 143B cells. Mice were infected i.p. with
5 3 105PFUs of either HSV-1 or VACV-gB. L. monocytogenes-gB DActA
expressing gB-8p was constructed as previously described (10) and grown
in brain heart infusion agar containing chloramphenicol (20 mg/ml). Prior
to infection, the bacteria were grown to log phase (OD6000.1), and mice
were injected i.v. with 1 3 106CFUs in 100 ml PBS.
Reagents and flow cytofluorometric analysis
The gB-8p:Kbtetramer was obtained from the National Institutes of Health
Tetramer Core Facility (Emory University, Atlanta, GA). mAbs anti-CD8a
(clone 53-6.7), anti-CD4 (RM4-5), anti-CD11a (2D7), anti-Vb10 (B21.5),
and anti-Vb8 (F23.1) were purchased from commercial sources. Flow
cytofluorometric data were acquired on the custom-made FACS LSRII
instrument equipped with four lasers, using Diva software (BD Bio-
sciences, San Jose, CA), and analysis was performed using FlowJo soft-
ware (TreeStar, Ashland, OR). At least 105cells were analyzed per sample,
with dead cells excluded by selective gating based on orthogonal and side
light scatter characteristics. Markers to score positive cells (events) were
set to delimit fluorescence higher than the highest fluorescence of un-
stained or control-stained cells.
Spleens were harvested from mice at the peak of infection and CD8+T cells
isolated using positive immunomagnetic selection of CD8+cells (Miltenyi
Biotec, Auburn, CA). Highly enriched CD8+T cells were stained with gB-
8p:Kbtetramers conjugated to streptavidin APC, anti–CD8a-PE Texas
Red, anti–CD4-FITC, and anti–Vb10-PE for 1 h and washed twice. Cells
were then transferred to sorting buffer, and CD8+CD42gB-8p:Kb+Vb10+
lymphocytes were isolated as single cells using the FACSAria cell sorter
system (BD Biosciences). Control wells without sorted cells were included
on every plate to identify any possible contamination.
Our RT-PCR protocol was adapted from that of Kedzierska et al. (14) and
Hamrouni et al. (18). Single cells with appropriate surface markers were
sorted directly into 96-well PCR plates containing 5 ml cDNA reaction mix.
The cDNA reaction mixture contained 0.25 ml Sensiscript reverse tran-
scriptase (Qiagen, Valencia, CA), 13 cDNA buffer (Qiagen), 0.5 mM 29-
deoxynucleoside 59-triphosphate (Qiagen), 100 mg/ml tRNA (Invitrogen,
Carlsbad, CA), 50 ng oligodT12–18(Invitrogen), 20 U RNAse Out (In-
vitrogen), and 0.1% TritonX-100 (Sigma-Aldrich, St. Louis, MO). cDNA
synthesis was performed immediately after sorting by incubating plates at
(Fisher Scientific, Malvern, PA), 200 uM each 29-deoxynucleoside 59-tri-
phosphate (Fisher Scientific), and 100 nM external sense Vb10 primer (59-
CTCAGCTCCACGTGGTCA-39). The PCR conditions for the first PCR
at 59˚C, and 45 s at 72˚C, ending with 5 min at 72˚C. A 1.0-ml aliquot of the
first-round PCR wasused for the second PCR reaction with the internalsense
Vb10 primer (59-GCAACTCATTGTAAACGAAACA-39) and internal anti-
sense Vb10 primer (59-CGAGGGTAGCCTTTTGTTTG-39). The second
PCR program began with 95˚C for 2 min, then 72˚C for 5 s, followed by 35
PCR products were resolved on a 2% agarose gel, purified with the MinElute
96 UF PCR purification kit (Qiagen), and sequenced with 12 pmol Vb10 se-
quencing primer (59-AGGCGCTTCTCACCTCAGTCTTC-39), using an
Analysis and Technology Core (University of Arizona, Tucson, AZ). The
internal sense primer was also used as a sequencing primer for mice infected
TCRb repertoire analysis
The gB-8p–specific CD8+TCRb repertoires were characterized by se-
quentially aligning each of the TCRb sequences with the Vb10 (TRBV4 in
the ImMunoGeneTics nomenclature) gene, the best match Jb gene, the
best match Db gene, and then identifying the CDR3b sequence. The
reference alleles for the Mus musculus germline TRB genes from the Im-
MunoGeneTics nomenclature (19) were used for the analysis of the TCRb
To assess whether the diversities of the TCRb repertoires specific for the
gB-8p-epitope differed among the HSV-1, L. monocytogenes-gB, and
VACV-gB infections, both the number of different amino acid sequence
clonotypes and Simpson’s diversity index (20) were used as measures of
clonotypic diversity. Simpson’s diversity index accounts for both the va-
riety of amino acid sequence clonotypes and their clone sizes, and ranges
in value from 0 (minimal diversity) to 1 (maximal diversity). To account
for the differences in the sample sizes obtained between mice, the TCRb
repertoire diversities were estimated as if 39 TCRb sequences had been
obtained for each sample. This estimation involved randomly drawing
from each sample a subset of 39 TCRb sequences and estimating the di-
versity of this subset. This process was repeated 10,000 times to produce
a distribution of TCRb repertoire diversities, from the median of which we
determined a diversity estimate for each sample (20). The diversity anal-
ysis was performed using Matlab (The Mathworks, Natick, MA).
The Journal of Immunology2959
8p–specific CD8+TCRb repertoires at day 6 postinfection were compared
among the groups of mice infected with HSV-1, L. monocytogenes-gB, and
VACV-gB, using a Kruskal-Wallis test and Dunn’s multiple comparison
in immune responses in mice in the three different infection groups were
compared using a nonparametric two-way ANOVA and Bonferroni postt-
ests (i.e., based on ranks). All statistical analyses were performed using
GraphPad Prism software (GraphPad, San Diego, CA).
Kinetics and magnitude of the gB-8p–specific CD8+T cell
responses in mice infected with HSV-1, L. monocytogenes-gB,
C57BL/6 mice is directed against the single immunodominant
gB-8p epitope (21). To determine whether the context of infection
alters the kinetics and magnitude of the CD8+T cell response, we
first monitored the expansion of gB-8p–specific CD8+T cells in
mice infected with HSV-1, L. monocytogenes-gB, and VACV-gB.
The dramatic increase of gB-8p–specific CD8+T cells was tracked
in the spleen. Although the overall kinetics of CD8+T cell ex-
pansion were similar among infections, the frequency and total
number of gB-8p–specific CD8+T cells were two to three times
higher in mice infected with HSV-1 and VACV-gB than in those
infected with L. monocytogenes-gB (Fig. 1). This quantitative
difference in clonal expansion between different vectors is most
likely explained by the attenuated nature of the Listeria vector
(L. monocytogenes-AcA-gB), which is expected to provide lower
stimulation of the immune system.
The gB-8p–specific TCRb repertoires from mice infected with
HSV-1, L. monocytogenes-gB, and VACV-gB exhibit similar
Vb segment and sequence compositions
To uncover any broad differences in the CD8+TCR repertoire among
including these from our laboratory, have documented significant Vb
(22–26). When we compared Vb use within gB-8p–specific CD8+
T cells from mice infected with HSV-1, L. monocytogenes-gB, and
VACV-gB, we observed a similar Vb bias in response to all three in-
fections at day 6 postinfection (Fig. 2). Although no significant dif-
ferences in Vb10 use of gB-8p–specific CD8+T cells were observed
between HSV-1 infection and either L. monocytogenes-gB or VACV-
gB infection (where 50.0%, 57.8%, and 55.4% of gB-8p–specific
CD8+T cells used Vb10 for L. monocytogenes-gB, VACV-gB, and
HSV-1, respectively), there was a small but statistically significant
difference in Vb10 use in mice infected with L. monocytogenes-gB
and Bonferroni posttest). We do not consider this difference bi-
ologically significant, given that Vb10 makes up an overwhelming
part of this response in each of the three responses. By contrast, Vb8
use was extremely similar among all three infections (i.e., 22.0%,
22.3%, and 22.3% of gB-8p–specific CD8+T cells for the HSV-1, L.
monocytogenes-gB, and VACV-gB infections, respectively). Overall,
Vb use profiles among infections were largely comparable. Thus,
despite major differences in the magnitude of the CD8+T cell re-
sponse, different intracellular pathogens (HSV-1, L. monocytogenes-
selecting widely different Vbelements.
For a more in-depth assessment of the composition of the TCRb
repertoires involved in the gB-8p–specific CD8+T cell responses in
HSV-1, L. monocytogenes-gB, and VACV-gB infections, the CDR3
portions of the TCRb repertoires using the Vb10 gene were se-
quenced. These data are summarized in Table I. Given the enormous
analysis was required to resolve these questions and conclusively
examine key factors that shape epitope-specific TCR repertoires. A
L. monocytogenes-gB, and VACV-gB infection groups. The HSV-1
infected with HSV-1, L. monocytogenes-gB, and VACV-gB. The kinetics
of the gB-8p–specific CD8+T cell responses was tracked in the spleen of
mice infected with HSV-1, L. monocytogenes-gB, and VACV-gB (A), and
the magnitude of the gB-8p–specific CD8+T cell responses was compared
for all three infections at day 6 postinfection (B). Significant differences in
the percentages of gB-8p–specific CD8+T cells at day 6 postinfection
among the L. monocytogenes-gB and both the HSV-1 and VACV-gB in-
fections were determined by a Kruskal-Wallis test. Results are represen-
tative of at least two separate experiments, with n $ 4 mice/group. pp ,
0.05, mean 6 SEM.
Profiles of gB-8p–specific CD8+T cell responses in mice
L. monocytogenes-gB, and VACV-gB infections. A comparison of the per-
centages of gB-8p–specific CD8+T cells using the Vb10 and Vb8 genes
among all three infections at day 6 postinfection. There were no significant
differences in the Vb10 or Vb8 gene use of gB-8p–specific CD8+T cells
between the HSV-1 infection and either the L. monocytogenes-gB or the
in the L. monocytogenes-gB infection was found to be significantly lower
than for the VACV-gB infection, using a nonparametric two-way ANOVA
and Bonferroni posttest. The graphs represent pooled results from two sep-
arate experiments, with n = 4 mice/group. pp , 0.01, mean 6 SEM.
2960CD8+TCR REPERTOIRE INDEPENDENT OF PATHOGEN CONTEXT
and L. monocytogenes-gB infection groups consisted of 10 mice
each, whereas the VACV-gB infection was studied in 6 mice. The
number of TCRb sequences per mouse varied between 39 and 82,
with an average of 61.8 TCRb sequences per mouse. Examples of
the gB-8p–specific Vb10+TCRb amino acid sequence repertoires
for one mouse from each of the HSV-1, L. monocytogenes-gB, and
VACV-gB infection groups are shown in Table II.
The compositions of the gB-8p–specific Vb10+TCRb reper-
toires for the three infections are summarized by the distributions
of CDR3b lengths (Fig. 3A; in this study, the CDR3b sequence is
inclusive of the conserved cysteine in the Vb region and the
conserved phenylalanine in the Jb region, as shown in Table II)
and Jb gene use (Fig. 3B) for each mouse. The variation in
CDR3b length and Jb gene use among mice in the different in-
fection groups was comparable to that among mice in the same
infection group. The gB-8p–specific Vb10+TCRb repertoires
were also pooled across all mice within each infection group, and
the proportions of TCRb clonotypes (ignoring the number of
copies of each TCRb sequence) with a particular CDR3b length
(Fig. 3C) and using a particular Jb gene (Fig. 3D) were compared
T cell responses in HSV-1, L. monocytogenes-gB, and VACV-gB infections
Summary of the data used to compare the Vb10+TCRb repertoires for the gB-8p–specific CD8+
HSV-1 L. monocytogenes-gB VACV-gB
No. of mice
No. of TCRb sequences across all mice
Range of no. of TCRb sequences per mouse
Mean no. of TCRb sequences per mouse
The Vb10+TCRb repertoires involved in gB-8p–specific CD8+T cell responses in one mouse each for the HSV-1, L. monocytogenes-gB, and
HSV-1: Mouse 6L. monocytogenes-gB: Mouse 5VACV-gB: Mouse 6
aWG doublets in the CDR3b are shown in bold.
The Journal of Immunology2961
among HSV-1, L. monocytogenes-gB, and VACV-gB infections.
The dominant length of the CDR3b amino acid sequences was 14,
with at least 58% of the pooled gB-8p–specific Vb10+TCRb
clonotypes for each infection having a CDR3b length of 14 aa.
CDR3b sequences of length 13 and 15 aa were also common,
constituting .14% of the gB-8p–specific Vb10+TCRb clono-
types for each of the three infections (Fig. 3C). Preferential use of
the Jb2 group of genes was found, with at least 95% of the gB-8p–
specific Vb10+TCRb clonotypes for each infection using Jb2
genes (Fig. 3D). This bias toward use of the Jb2 group of genes is
consistent with observations in previous studies of the HSV-1 gB-
8p–specific TCRb repertoire (22, 23, 27).
This comparison demonstrates that the gB-8p–specific TCRb
repertoires involved in CD8+T cell responses in infections with the
live recombinant vectors L. monocytogenes-gB andVACV-gB have
infection in which the gB-8p epitope is encountered has little in-
fluence on the composition of the responding TCRb repertoire.
Infection with different pathogens does not alter the diversity of
the TCR repertoire responding to the gB-8p epitope
The diversity of TCRs responding to anepitope is thought tobe one
determinant of the ease of immune escape by viruses. Diversity is
often measured as the number of different TCRs seen in an animal.
However, this does not take into account either the clonal domi-
nance among these TCRs (i.e., if there are 10 different TCRs, do
they each make up 10% of the response, or does one make up 91%,
and the other 1% each?) or the number of TCRs sequenced. To
directly address whether the type ofinfection inwhich an epitopeis
encountered influences the sequence diversity of the epitope-
specific CD8+T cell response, the clonotypic diversities of the
TCRb repertoires responding to the gB-8p epitope were compared
among HSV-1, L. monocytogenes-gB, and VACV-gB infections.
The diversities of the TCRb repertoires were estimated for each
mouse, as if each sample was of the same size, using both the
number of different amino acid sequence clonotypes and Simp-
son’s diversity index as diversity measures. The latter diversity
measure accounts for both the variety of different TCRb clono-
types and the clonal dominance hierarchy and varies in value
between 0 and 1, which reflects minimal and maximal diversity,
respectively (20). The gB-8p–specific TCRb repertoires were very
diverse in all mice in all three infection groups, with at least 19
different clonotypes and Simpson’s diversity indices .0.92 (Fig.
4A, 4B). For the HSV-1, L. monocytogenes-gB, and VACV-gB
infections, the median numbers of clonotypes were 24.5, 28, and
27, and the median Simpson’s diversity indices were 0.96, 0.98,
and 0.97, respectively. No significant differences were seen in the
diversities of the gB-8p–specific TCRb repertoires among the
HSV-1, L. monocytogenes-gB, and VACV-gB infections.
Vb10+TCRb repertoires involved in CD8+T cell re-
the gB-8p–specific Vb10+TCRb repertoires in each
length refers in this study to the length of the sequence
conserved phenylalanine in the Jb region, as shown in
Table II. The mice in each infection group are shown in
numerical order of their mouse numbers (i.e., mouse 1,
Vb10+TCRb clonotypes for all mice in each infection
group were pooled to compare the proportion of gB-8p–
1, L. monocytogenes-gB, and VACV-gB infections.
Comparison of the compositions of the
2962 CD8+TCR REPERTOIRE INDEPENDENT OF PATHOGEN CONTEXT
important in an epitope-specific T cell response, because conserved
patterns of amino acid use in CDR3 can indicate a restricted TCR
epitope, preferential use of the Db2 gene in a single reading frame
encodes a tryptophan-glycine (WG) doublet in CDR3b positions 3
and 4 [using the Chothia definition (29)], resulting in a relatively
conserved junctional sequence (27). The example gB-8p–specific
TCRb repertoires shown in Table II suggest that the WG doublet is
[where positions 6 and 7 of the CDR3b sequences in Table II cor-
respond to Chothia CDR3b positions 3 and 4 (29)]. Our in-
vestigation of the prevalence of the WG doublet in CDR3b
8p–specific TCRb repertoires found that at least 88% of both the
TCRb repertoire, and the TCRb clonotypes, in all mice exhibited
the WG signature, regardless of the infection (Fig. 4C, 4D).
Moreover, no significant differences were observed in the preva-
lence of the WG doublet among the HSV-1, L. monocytogenes-gB,
and VACV-gB infection groups. In all three infections, this WG
previous studies of TCRb repertoires responding to the HSV-1 gB
The fundamental question we wished to address in this paper was
repertoire simplybychangingthevector usedtodelivertheepitope.
We addressed this question by comparing the TCRb repertoires
involved in CD8+T cell responses to a specific immunodominant
epitope in mice immunized withHSV-1, L. monocytogenes-gB, and
from mice infected with HSV-1 and live recombinant vectors
L. monocytogenes-gB and VACV-gB all have similar diversities,
suggesting that the nature of infection in which the gB epitope was
encountered has no significant influence on CD8 T cell repertoire
diversity. These conclusions were established on the basis of large-
scale analyses demonstrating the following results: 1) Vb and Jb
gene use and CDR3b lengths were similar among all infections; 2)
no significant differences in TCRb clonotypic diversity were de-
tected among all infections; and 3) no significant differences in the
It was surprising that the diversity in CD8+TCRb repertoires re-
sponding to a particular epitope was not altered among infections
with large DNA viruses (VACV-gB, HSV-1) that generate robust
expansion of CD8+T cells and an attenuated Gram-positive bacte-
nearby cells and results in a more diminished response. The context
of infection among VACV-gB, HSV-1, and L. monocytogenes-gB
may also differ in their levels of cytopathicity, infected cell types,
inflammatory cytokines, and Ag kinetics. Furthermore, there are
processing of Ags derived from bacterial versus viral vectors (30–
32), yet none of these dissimilarities resulted in a detectable differ-
ence in the diversity of responding CD8+T cells.
Given that in our experimental system, diversity of the
responding CD8 T-cells was not influenced by the microbial
context of the pathogen under widely different conditions, it is
important to highlight a few key differences between the report of
Malherbe et al. (8), in which differences in CD4+T cell repertoire
were presented, and our current study. First, previous reports in-
dicate that different priming requirements may be optimally re-
quired to activate CD4+and CD8+T cells. Whereas CD8+T cells
require just a brief episode of stimulation (33–35), CD4+T cells
require a more prolonged period of Ag stimulation (36, 37) which
may result in different levels of sensitivity in the clonal selection
process. Second, it is likely that more redundancy is present in the
immune response initiated with live infectious vectors compared
with peptide/protein immunizations. The cytokine milieu can
provide important signals involved in CD8+T cell expansion, but
some of these signals (IL-12 and type I IFN) have been shown to
monocytogenes-gB, and VACV-gB infections. TCRb clonotypic diversity was measured using both the number of different TCRb clonotypes (A) and
Simpson’s diversity index (B). Simpson’s diversity index accounts for both the number of different TCRb clonotypes and the number of copies of each
clonotype (i.e., the clonal dominance hierarchy) and varies between 0 (minimal diversity) and 1 (maximal diversity). The TCRb diversities were estimated
for all samples having an equal sample size of 39 TCRb sequences. The diversity of the CDRb sequences was considered by determining the prevalence of
the WG doublet in CDR3b positions 6 and 7, as shown in Table II [which correspond to CDR3 positions 3 and 4 using the Chothia definition (29)]. Shown
are the percentages of the TCRb clonotypes per mouse (C) and the percentages of the TCRb repertoires per mouse (including the number of copies of the
clonotypes) (D) that feature a WG doublet in CDR3b positions 6 and 7. A Kruskal-Wallis test was used to determine that there were no significant
differences in TCRb repertoire diversity among the HSV-1, L. monocytogenes-gB, and VACV-gB infections.
Comparison of the diversities of the Vb10+TCRb repertoires involved in CD8+T cell responses to the gB-8p epitope in HSV-1, L.
The Journal of Immunology 2963
a greater number of redundant signals are present in bacterial and
viral infections (compared with adjuvants), leading to minimization
of differences in the inflammatory environments among HSV-1,
between the findings of Malherbe et al. (8) and our present study
the TCR repertoire specific for the I-Ek–restricted PCC94–103epitope
is narrow and public, the repertoire specific for the Kb-restricted
gB498–505epitope is substantially more diverse, with less inter-
individual sharing of TCR clonotypes observed. It would be in-
teresting to examine all of these issues more closely and determine
whether the clonal composition of gB-8p–specific clonotypes could
be altered with different proteinvaccination strategies, or whether I-
Ek–restricted PCC94–103–specific T cells could be differentially
another potential difference between the Malherbe study and our
analysis is the number of animals and sequences analyzed. The for-
mer analyzed three mice per group, with ,20 sequences per animal,
and the results were pooled for the purposes of analysis. Our study
included larger groups (6–10 mice), with many more sequences per
mice, instead of the pooled repertoire, and thus avoided the potential
problem of a “rogue mouse” biasing the overall repertoire. It will be
important to repeat studies of TCR diversity in the CD8+and CD4+
resolve the differences between these studies.
Regardless of vaccine strategy, the fundamental unit of T cell
responses is the individual clonotype, and T cell immunity must be
described in these terms if we are to understand the exact structure
of a T cell response that is essential for controlling acute and
chronic pathogens. In a recent report, Price et al. (39) demonstrated
that certain CD8+TCR repertoire features could predict vaccine
efficacy in SIV-challenged rhesus macaques. Understanding the
vaccine strategies that can generate and maintain these ideal
repertoires is of obvious importance, but this requires us to un-
derstand whether these features are inherent to specific epitopes or
whether they can be altered by different vaccination approaches.
Our data indicate that equally diverse CD8+TCR repertoires can
be generated by widely different live vaccine vectors. Thus, many
attributes of the TCR repertoire may not be easily manipulated by
vaccination, and finding ideal epitopes may be a more productive
goal for efficacious immunizations.
We thank members of the Nikolich and Davenport laboratories for help and
stimulating discussion and the National Institutes of Health Tetramer Facil-
ity at Emory University for expert tetramer production.
The authors have no financial conflicts of interest.
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The Journal of Immunology 2965