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*
Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322; and
Unite de Biologie Moleculaire du Gene Institut Pasteur, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs, as well as complementarity-determining region 3
length distribution analysis of TCR V transcripts from cells sorted
according to tetramer binding. Following individual mice over time, we
found identity between primary effector and memory TCR repertoires for
each of three immunodominant epitopes from lymphocytic choriomeningitis
virus. During secondary responses, we found quantitative changes in
epitope-specific T cell hierarchies but little evidence for changes in
V usage or complementarity-determining region 3 length distributions
within epitope-specific populations. We conclude that 1) selection of
memory T cell populations is stochastic and not determined by a
distinct step of clonal selection necessary for survival from the acute
responding population, and 2) maturation of the T cell repertoire
during secondary lymphocytic choriomeningitis virus infection alters
the relative magnitudes of epitope-specific responses but does not
significantly modify the repertoire of T cells responding to a given
epitope. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD8+ T cell recognition of infected cells is
accomplished by the interaction of clonally distributed TCRs on
effector cells with the trimolecular complex of viral peptide/MHC class
I (MHC I)3/2-microglobulin on
infected cells (12, 13). The TCR is a disulfide-linked,
membrane-bound heterodimer of 
- and -chains, each the result of
unique somatic rearrangements in variable (V), joining (J), and, in the
case of the -chain, diversity (D) segments, during T cell
development and maturation (14, 15). These are in turn
joined to one of a limited number of functionally similar constant (C)
regions. The broad diversity of the naive TCR repertoire is a direct
result of this recombination process along with random incorporation of
junctional nucleotides. Further diversity is generated by the
combinatorial pairing of different - and -chains
(16). Similar to what is seen in Ig proteins, the
diversity of the TCR is focused into three complementarity-determining
regions (CDRs), termed CDR1, CDR2, and CDR3. The CDR1 and CDR2 are
encoded in the germline V segment genes, whereas the CDR3
encompasses the V-J, or in the case of the -chain, the
V-(D)n-J junction (14). Thus, the
most diverse portion of the TCR is the CDR3 of the -chain.
Functional (17, 18, 19) as well as recent crystallographic
data (20) have indicated that the CDR3 is centered over
the peptide/MHC I complex and makes direct contact with the presented
peptide, whereas the CDR1 and CDR2 are situated peripherally to the
CDR3 and make more extensive contact with the MHC. Nevertheless,
specificity of T cells for a particular MHC/peptide combination is, at
least in part, determined by contacts made by the CDR1 and CDR2, and
consequently, the V gene segment used (15). Analysis of
the specificity of Ag recognition has shown restricted CDR3 lengths
within particular V-expressing subpopulations of responding cells,
suggesting that the length of this region is a major determinant of TCR
specificity (2, 21, 22, 23).
Studies of TCR repertoires within Ag-specific populations have shown that some populations of epitope-specific cells show restricted TCR diversity (2, 24, 25), whereas others are of a considerably broader distribution (26, 27). However, many of these analyses have relied upon in vitro expansion of Ag-specific subsets of cells, a process prone to experimental bias. TCR repertoires that have been analyzed ex vivo are limited to systems containing one immunodominant epitope possessing restricted diversity (24, 25). The relationship between primary Ag-specific effector cells and memory cells in these systems supports a stochastic selection process in which the repertoire of the memory pool directly reflects that which is present in the effector population (22, 24, 25, 28). However, recent evidence suggests that, upon secondary exposure to the same antigenic determinants, there is a selective expansion or "focusing" of Ag-specific cells both in terms of diversity of their repertoires as well as affinity of the TCR for its ligand (29, 30). This focusing can be viewed from at least two different levels. Populations specific for one epitope may be privileged to expand over others (4), whereas within a given epitope-specific population, there may be preferential expansion of certain TCR subpopulations (29).
We have performed direct ex vivo analysis of CD8+
T cell populations specific for three concurrent immunodominant
epitopes generated during infection of C57BL/6J
(H-2b) mice with lymphocytic choriomeningitis
virus (LCMV) (4, 5). Two of these, NP396-404 and GP33-41,
are presented in the context of the H-2Db MHC I
molecule. The third immunodominant epitope, GP34-41, although a 1-aa
truncation of the Db-restricted GP33-41 epitope,
is presented by H-2Kb (31). Using
soluble MHC I tetramer reagents, as well as a panel of V-specific
Abs, we have analyzed the TCR V usage of these three
epitope-specific populations. Sorting specific cells, we have also
analyzed the CDR3 distribution of each of these populations. Due to the
high degree of mouse-to-mouse variation in V usage found in this and
other studies (28, 32, 33), we also followed the TCR
repertoire of LCMV epitope-specific cells in individual mice within
primary effector, memory cell, and secondary effector populations to
more accurately determine the relationship between these
populations.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adult C57BL/6J mice (B6, H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were housed in American Association for Accreditation of Laboratory Animal Care (AAALAC) accredited facilities. For primary challenge and memory analyses, mice were infected i.p. with 2 x 105 PFU of the Armstrong strain of LCMV and were used at the indicated time post infection. For secondary challenge analyses, immune B6 mice primed >6 mo previously with LCMV Armstrong were infected i.v. with 2 x 106 PFU of the clone 13 strain of LCMV and used 4–5 days postinfection. Virus stocks were grown and maintained as previously described (34).
Cells and flow cytometry
Abs used in this study were purchased from PharMingen (San
Diego, CA) with the exception of the anti-CD8 Ab (clone CT-CD8a;
Caltag, Burlingame, CA) used to reveal
KbGP34-41-specific cells. MHC I tetramers were
prepared and refolded with the appropriate peptide and
2-microglobulin as previously described
(35). Single cell suspensions of splenocytes were prepared
as previously described (4). PBMC were separated on
Histopaque-1077 (Sigma, St. Louis, MO) in accordance with the
manufacturer’s recommendations. For analysis of V usage by
epitope-specific populations, 
106 cells were
stained with the indicated reagents in FACS buffer (PBS containing 2%
BSA and 0.2% sodium azide) for 30 min at 4°C, followed by three
washes in FACS buffer. Samples were acquired on a FACScalibur flow
cytometer (Becton Dickinson, San Jose, CA). For sorting purposes,
2 x 107 splenocytes were incubated in 2
ml RPMI 1640 medium supplemented with 1% FCS with the appropriate
reagents for 30 min at 4°C. Cells were washed once and immediately
sorted on a FACSvantage flow cytometer (Becton Dickinson). Specificity
of sorted populations was confirmed by postsort flow cytometric
analysis as well as measurement of IFN-
production in response to
peptide epitopes by enzyme-linked immunospot assays.
Assays for IFN- production
Enzyme-linked immunospot assays were performed as previously
described (4). Microfiltration plates (Millipore, Bedford.
MA) were coated with purified anti-IFN- Ab (clone R4-6A2). Cells
were cultured with or without the appropriate peptide for 36 h at
37°C with 5 x 105 -irradiated naive
syngeneic splenocytes as stimulator cells. Following this period, cells
were washed off and incubated with a second, biotinylated
anti-IFN- Ab (clone XMG1.2) overnight at 4°C. A secondary
incubation step was then performed with the addition of
streptavidin-conjugated to HRP (Vector Laboratories, Burlingame, CA).
Spots were developed by the addition of substrate buffer (0.03%
3-amino-9-ethyl-carbazole, 0.015% hydrogen peroxide
(H2O2) in 0.1 M sodium
acetate). The spots produced by the colored, insoluble cleavage product
of 0.03% 3-amino-9-ethyl-carbazole were then counted, and the
frequencies of epitope-specific cells were determined. Measurement of
intracellular IFN- in response to synthetic viral epitopes was
performed as previously described (4).
Immunoscope analysis
RNA from sorted cells was extracted using guanidinium
isothiocyanate disruption followed by phenol/chloroform extraction and
ethanol precipitation (36). First strand cDNA synthesis
was performed as previously described (25). PCR
amplifications using 24 mouse V-specific primers (25)
as well as a single mouse C primer were performed in 96-well
microtiter plates (37). Following amplification, an
eight-round run-off linear amplification step was performed using an
internal 6-carboxyfluorescein-labeled C primer. Products and m.w.
standards were run on a 4.0% polyacrylamide denaturing gel on an
Applied Biosystems (Foster City, CA) 377 automated sequencer. Peak
intensities were extracted using Applied Biosystems sequence analysis
3.0 software and analyzed using Immunoscope 1.0 software
(37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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usage of LCMV-specific primary effector cells
Adult B6 mice infected with the Armstrong strain of LCMV exhibit a
dramatic increase (>10-fold) in the total number of activated
CD8+ T cells during the first 8 days following
infection (4). During this expansion, the number of
CD8+ cells within each of the V-expressing
populations also increased dramatically per spleen (Fig. 1
). It has recently been shown that the
large expansion of CD8+ T cells seen during LCMV
infection is primarily due to an increase in the number of
virus-specific CD8+ T cells (4, 5).
Considering the number of epitope specificities represented in these
cells (4, 31), the antiviral response includes
CD8+ T cells using TCR that encompass virtually
all of the expressed V gene segments.
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20 and 15%, respectively, of all activated
CD8+ T cells at the height of the immune
response. However, functional analysis of this specificity is
confounded by the presentation of the GP34-41 peptide in the context of
H-2Kb, accounting for roughly half of the IFN-
response elicited by the GP33-41 peptide (J. D. Altman, manuscript
in preparation; Ref. 38).
We analyzed the TCR-V repertoire of CD8+ T
cells specific for each of these three epitopes by flow cytometry,
costaining CD8+ T cells with
Db and Kb MHC I tetramers
(DbNP396-404, DbGP33-41,
KbGP34-41), and a panel of V-specific Abs. A
representative analysis of V/MHC I tetramer costains of
CD8+ T cells from an individual LCMV-infected
mouse is shown in Fig. 2. Abs to V
regions not indicated in the figure were either not available at the
time of analysis or did not contribute to the epitope-specific
populations examined; with the indicated reagents, we were able to
detect 80–85% of the total CD8+ population.
Although we did not attempt to determine the relative distribution of
the remaining 15–20% of CD8+ T cells,
immunoscope analysis of TCR transcripts from tetramer-sorted cells
(described below) indicates that this population did not contain a
significant fraction of LCMV-specific cells. There were no significant
differences in the number of CD8+ cells that
specifically bound the MHC I tetramers in the presence or absence of
any of the anti-V Abs used or in assays measuring functional
responses to synthetic viral epitopes (24, 25).
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usage within these three epitope-specific
populations from five representative individual mice at the peak of the
primary immune response to LCMV is shown in Fig. 3. Consistent with a previous report
(28), a portion of each of the epitope-specific
populations consisted of V8.1,8.2+
CD8+ cells (Fig. 3). The magnitude of this
contribution to each epitope-specific populations varied not only
between epitopes (comparing vertically, Fig. 3, A, F, and
K; B, G, and L; C, H, and
M; D, I, and N; E, J, and
O), but also between mice (comparing horizontally, Fig. 3, A–E, F–J, and K–O). In addition to
V8.1,8.2+ cells, conserved "signature"
V usage profiles were seen for each of the epitopes examined.
Characteristically, usage of V6 and V9 gene segments was observed
by DbNP396-404-specific cells (Fig. 3, A–E) and, with V8.1,8.2+ cells,
comprised greater than two-thirds of the entire
DbNP396-404-specific repertoire. Commitment of
CD8+ T cells within the
V8.1,8.2+ population to this response was
high, as 30% of all V8.1,8.2+ cells were
specific for the DbNP396-404 epitope. Similarly,
there was a high level of commitment within V6- and V9-expressing
CD8+ T cells to the
DbNP396-404-specific response as 25% of these
cells were specific for this epitope. Commitment within other
V+ lineages to the
DbNP396-404 response was low.
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8.1,8.2 or V7 Abs and comprised 50% of the entire
epitope-specific population (Fig. 3
, F–J). There was also
variable recruitment of V6, V9, V10, V11, or V13
positive cells, with these subpopulations contributing 30% of the
total DbGP33-41-specific response. Similar to
what was seen with DbNP396-404-specific cells,
commitment of V7-expressing cells to the
DbGP33-41 response was high, as >20% of all
CD8+V7+ cells were
specific for this epitope. In contrast, although a large number of
V8.1,8.2+ cells were recruited into the
DbGP33-41 response, <6% of
CD8+V8.1,8.2+ cells were
specific for this epitope. Commitment within V populations
constituting minor portions of the DbGP33-41
response was also low when compared with that seen for V7-expressing
cells.
A much more restricted pattern of V usage was seen in
CD8+ T cells specific for the
KbGP34-41 epitope (Fig. 3, K–O). In
this case, V8.1,8.2+CD8+
T cells represented a minor portion of the response, whereas the major
V gene segment used, V11, constituted >20% of the
KbGP34-41-specific population (Fig. 3, K–O). Commitment within V11-expressing cells to the
KbGP34-41 response was high, as 25% were
specific for this epitope. However, similar to
DbGP33-41-specific cells, there was a low
commitment within the V8.1,8.2+ population to
this response. Importantly, the V usage profiles between the
DbGP33-41 and
KbGP34-41-specific CD8+
were drastically different, providing further evidence of the distinct
nature of these two populations (Fig. 3, F–J,
K–O).
For each of the epitopes examined, many of the mice recruited T cells
expressing V regions that were variably observed. These included
V7 and V13 in DbNP396-404-specific
populations; V6, 9, 10, 11, and 13 within
DbGP33-41-specific populations; and V10,12,
and 13 in KbGP34-41-specific cells. The most
diversity in V gene segment use was seen in
DbGP33-41-specific populations (Fig. 3, F–J). Preliminary analysis also indicates significant
semiconservative expansion of V8.3-expressing cells within
DbGP33-41-specific populations as well as V5,
V8, V10, and V14 within populations specific for a another
LCMV epitope, DbGP276-286. Taken together, these
analyses account for the broad expansion in virtually all of the
different TCR-V CD8+ T cell populations during
the primary response to LCMV (Fig. 1A).
Immunoscope analysis of LCMV-specific primary effector cells
An alternative method for the elucidation of TCR repertoires has
been the RT-PCR-based analysis of CDR3 length distributions known as
immunoscope or spectratyping analysis (23, 25, 28, 39).
Primarily, this approach has been used to identify responses based on
the skewing of CDR3 length distributions within bulk T
cell-V-expressing subpopulations due to expansions of restricted
precursors. We modified this approach by first sorting Ag-specific
populations, thereby reducing "background" peaks. This enabled us
to further resolve each of the signature responses even within small
populations using a broad range of V gene segments. In addition,
this method allowed us to identify Ag-specific expansions within V
subpopulations for which there were no commercially available Abs at
the time of analysis. Using this approach, we found systematic usage of
certain CDR3 lengths within each of the signature V responses
previously identified (Fig. 4, Table I). For the conserved V responses
within DbNP396-404-specific populations, there
was a systematic usage of TCR having CDR3 lengths of 9 aa by
V6+ cells, 11 aa by
V7+, 9 aa by V8.1+,
and 9 and 10 aa by V9+ cells. Within
DbGP33-41-specific populations, there was
conserved usage of TCR with CDR3 lengths of 9 aa by
V7+, 8 and 9 aa by
V8.1+, and 8 aa by
V8.3+ cells.
KbGP34-41-specific populations reproducibly used
TCR having CDR3 lengths of 9 aa by V8.1+, 10
or 11 aa by V11+, and 8 aa by
V12+ cells. In addition to these conserved
CDR3 lengths, other CDR3 length responses were sporadically observed in
individual mice as shown in Table I. Within variable V responses
identified by V/MHC I tetramer costains, no predictable pattern of
CDR3 length usage was observed. CDR3 responses were similar between
primary effector, memory cell, and secondary effector populations (data
not shown).
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usage within LCMV-specific memory cells
Following the initial expansion of the immune response and
clearance of the virus, a death phase ensues in which >90% of
activated CD8+ cells undergo apoptosis, leaving
behind a stable pool of memory cells (Fig. 5) (4). The distribution of
V usage within bulk CD8+ T cells from immune
mice showed no differences when compared with naive animals (Fig. 1B). This is due to both the number of epitopes at which the
CD8+ response is directed as well as the
diversity in V usage within each of those epitope-specific
populations, effectively diluting the memory scar over a larger
CD8+ T cell repertoire. Approximately 3–5% of
CD8+ cells in immune mice were specific for the
DbNP396-404 epitope, whereas the
DbGP33-41- and
KbGP34-41-specific cells each constituted
1–2% of CD8+ cells. Our analysis of LCMV
epitope-specific CD8+ cells showed the same mean
distribution of V usage between primary effector and memory
populations (Fig. 6A and
D; B and E; and C and
F) In addition, the signature V responses that were seen
during primary responses were always recapitulated in memory
populations. However, there was still a high degree of variation in the
magnitude of these signature responses between mice (Fig. 6, A and D; B and E; and
C and F).
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usage within secondary effector cellsTo examine the Ag-specific T cell repertoires during a recall response, mice that had been primed i.p. with 2 x 105 PFU of LCMV Armstrong 3–6 mo earlier were challenged i.v. with 2 x 106 PFU of LCMV clone 13. In contrast to well established models for the study of recall T cell responses to influenza A, in the LCMV model it is not possible to obtain reassortment viruses that share T cell epitopes but not determinants for neutralizing Abs. Therefore, the dose and route for the challenge were chosen to permit establishment of infection in mice that had substantial cellular and humoral immunological memory to LCMV. Although we did not attempt to account for the possible influence that circulating virus-specific Ab might have on the T cell repertoires during the recall response, experiments in CD4 knockout mice, which lack an Ab response, revealed very similar LCMV-specific T cell repertoires compared with wild-type mice (data not shown).
During secondary infection with LCMV there was a preferential expansion
of DbNP396-404-specific
CD8+ T cells when compared with either the
DbGP33-41 or the KbGP34-41
epitopes (Fig. 5). Expansion of
DbNP396-404-specific cells was observed to be
30-fold, whereas expansion of the KbGP34-41-
and DbGP33-41-specific populations showed 10-
and 5-fold expansion. Thus the ratio of epitope-specific cells changed
from 1.5:1:1
(DbNP396-404:DbGP33-41:KbGP34-41)
during primary responses and memory phase to 6:1:2 during secondary
responses. Recent studies on the relationship of secondary to primary
effector pools have shown that there is a narrowing of the TCR
repertoire due to selective expansion of subpopulations of cells
(29, 30). Total CD8+ T cell
expansion of V subpopulations upon secondary expansion was similar
to that seen during primary expansion (Fig. 1A). However,
there appeared to be privileged expansion of V6 and
V9 (two of the TCR-V represented in
DbNP396-404-specific populations) as well as
V10+ cells, part of the
DbGP276-286 response, which also becomes more
prominent during secondary responses (Fig. 1B). Thus the
differences that were observed in the hierarchy of
CD8+ V+ subpopulations
were attributable to the preferential expansion of
DbNP396-404- or
DbGP276-286-specific populations.
Upon comparison of many mice, we saw evidence that within the
DbNP396-404-specific population, there was a
minor preferential expansion of V8.1,8.2+
cells when compared with V6- or V9-expressing populations (Fig. 6G). Additionally, within the
KbGP34-41-specific population, there appeared to
be a preferential expansion of V11-expressing cells (Fig. 6I) and, to a lesser degree, V7-expressing cells within
the DbGP33-41-specific population (Fig. 6H), over V8.1,8.2+ cells. However,
due to the high degree of diversity seen in the magnitude of these
signature responses during both primary and secondary expansions, it is
impossible to determine whether the observed differences in V usage
in these rechallenged mice were originally skewed within individual
mice during the primary effector and memory phases.
Longitudinal analysis of V usage
Experiments using both PBMC and splenocytes from individual mice
showed no differences in the repertoires of Ag-specific cells
represented within these compartments (data not shown). Therefore, to
more accurately determine the relationship between primary effector,
memory, and secondary effector TCR repertoires, we followed
LCMV-specific CD8+ T cells present in PBMC of
individual mice over time. As the limited blood volume available from
each mouse permitted us to perform only six FACS stains, we were able
to determine the V repertoire for only one specificity per mouse,
where each stain contained one FITC- and one PE-labeled V Ab. Shown
in Fig. 7 are the V usage profiles for
each of the epitopes examined of five individual mice during primary
and secondary responses to challenge with LCMV, as well as the mean of
two memory phase time points. It is obvious from this analysis that
within DbNP396-404- (Fig. 7A),
DbGP33-41- (Fig. 7B), or
KbGP34-41- (Fig. 7C) specific
populations, there is near identity in the V usage profile between
primary effector and memory time points, supporting a model of
stochastic selection for the memory pool. Minor evidence was seen for
selection during recall responses to LCMV infection within
DbGP33-41- or
KbGP34-41-specific populations in terms of V
usage (Fig. 7, B and C). However, differences
were observed in the V usage profiles of
DbNP396-404-specific cells analyzed during
secondary challenges with LCMV. This focusing of the repertoire was
most apparent in mice in which the primary response had a broad
distribution of V usage (Fig. 7, A, 3 and 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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usage comprising 60–100% of the total
specific response. For each of the Db- and
Kb-restricted LCMV epitopes there was prevalent
usage of V8.1 gene segments among Ag-specific populations. This is
due, at least in part, to the large proportion of
CD8+ T cells in a naive mouse repertoire using
V8.1 or V8.2 (up to 25% of all CD8+ T
cells). This increases the probability of these V gene segments being
used by specific precursors. However, this does not adequately explain
this prominence, as there is a similar frequency of
V5.1,5.2+ cells in naive mice, but these cells
are not part of the epitope responses analyzed here. In addition to the
V8.1 response, DbNP396-404-specific
populations also reproducibly contained cells using V6 and V9.
For DbGP33-41-specific cells, the signature
response always contained V8.1 and V7 using cells, whereas
KbGP34-41-specific populations always contained
V11- and V8.1-expressing cells. These conserved responses are
likely due to interactions involving the CDR1 and CDR2 germline-encoded
regions within each V segment, providing a decreased stringency or
increased probability of specificity for each of the particular
MHC/peptide combinations (13). In addition to conserved
patterns of V usage within these epitope-specific populations, there
were also conserved CDR3 lengths used within each conserved V
subpopulation. The sum of these signature responses accounted for
roughly 60% of each of the epitope-specific populations.
There was considerable diversity in both the magnitude of the signature
responses as well as in the V usage and CDR3 lengths within the
remaining sporadic portion of the Ag-specific populations. The
variability in the sporadic responses most likely reflects low
frequencies of recruitable precursors. In such a model, the probability
of having an Ag-specific cell present in the naive pool and in the
correct environment to expand in response to an Ag is near some finite
threshold required for recruitment into the response. Because there is
a distribution of CDR3 lengths within any V+
population in the naive pool, the absolute number of cells using a
particular V-CDR3 combination can be limiting. Likewise, a more
stringent CDR3 sequence requirement can select for a low number of
precursors eligible for a given sporadic response. When the frequency
of such rare recruitable precursors falls below one per mouse, the
corresponding response will not be observed in all individuals and
might even only be sporadically observed. Under an ergodic hypothesis
of repertoire selection (40), the naive repertoires found
in different mice at any given time are equivalent to the naive
repertoires found within a single mouse at different time points. The
proportion of individuals undergoing a given sporadic response is a
measurement of the probability of the presence of the corresponding
precursors in a normal naive T cell mouse repertoire.
Prior analyses of a combined epitope repertoire using this system has
led others to postulate that it is "impossible" to compare the
repertoires of different mice taken during different times following
infection (28). This was based upon analysis of the
V8.1 response to LCMV,which contains many different specificities.
We show here that, in fact, when we deconvolute the V8.1 response,
there are predictable repertoires that emerge and are comparable. In
addition, as we have directly shown that V8.1+
T cells are recruited into the responses specific for each of at least
four epitopes, the analysis of the CDR3 length distribution for V8.1
from LCMV-infected mice must be performed on an epitope-specific basis
as with MHC I tetramer-sorted populations shown here. This becomes
especially important during chronic viral infections in which there are
dramatic changes in the hierarchy of epitope-specific populations.
We have also shown that the repertoires of the CTL populations present
within the primary response and memory pool are similar. This further
supports a stochastic model for selection of the memory pool (21, 22, 24, 25, 28, 32). This was true both when looking at the sum
of many mice or when comparing repertoires of effector and memory
CD8+ cells from individual mice. The small
differences seen between the V usage of effector and memory pools
from individual mice are within limits of experimental incertitude and
could be due to constant errors being applied to populations of
decreasingly small size.
During secondary recall responses, there was a selective expansion of DbNP396-404-specific cells, followed by KbGP34-41-, and DbGP33-41-specific populations. The change in the ratio of these populations as compared with the primary response is likely due to a difference in the kinetics of epitope presentation. Because there is a greatly reduced lag phase before the secondary response, cells responding first will be expanded preferentially. During the replicative cycle of LCMV, the nucleoprotein is produced first and is abundantly present in the cytoplasm of infected cells. Therefore, cells specific for nucleoprotein epitopes will be privileged to expand and kill infected cells, hampering secondary responses to other epitopes. This does not adequately explain the differential expansion of KbGP34-41- and DbGP33-41-specific cells as these overlapping epitopes are derived from the same portion of the glycoprotein. However, there may be differences in the amount or kinetics of presentation of these different epitopes due to differences in the way each is processed and presented.
Minor differences were observed in the V usage profiles of
DbNP396-404-specific cells expanded during
secondary responses when compared with primary effector or memory
pools. However, this was not the case for either the
DbGP33-41- or
KbGP34-41-specific populations. It is possible
that the strong antigenic challenge administered in this case was
sufficient to recruit the entire memory pool for these epitopes.
However, it is probable that the lesser expansion observed in these
populations during secondary responses minimized any observable
selection events between primary and secondary effector repertoires.
Previous reports comparing primary and secondary effector populations
have shown conflicting results, with some showing dramatic focusing of
the secondary repertoire due to expansion of only a portion of the
memory pool (11, 29, 30), whereas others have shown that
there were little or no differences between these populations
(22, 41, 42). It is possible that during the LCMV immune
response, most of the selection for high affinity TCR has taken place
during the primary response. Thus, we would not be able to detect
differences between primary and secondary repertoires in this
system.
It is still unclear whether there is any immunologic benefit gained from focusing of T cell responses. Maturation of B cell responses, with associated changes in the affinity of Ig molecules, occurs during the initial response when Ag becomes limiting. Thus, following each response there is an improvement in the functional ability of Ig molecules to bind and neutralize virus particles preventing infection. However, the results presented here show that, regardless of the diversity of the T cell response, the repertoire of memory cells reflects that of the preceding effector population. Selection of T cell populations occurs during periods of expansion when Ag is not limiting, so pathogens are able to establish themselves before the selection event. The relative importance of selection within T cell responses needs to be determined.
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Acknowledgments |
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Footnotes |
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2 J.N.B. and D.J.D.S. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. John Altman, Department of Microbiology and Immunology, Emory University Vaccine Center at Yerkes, 954 Gatewood Road, Atlanta GA 30329.
4 Abbreviations used in this paper: MHC I, MHC class I; CDR, complementarity-determining region; LCMV, lymphocytic choriomeningitis virus.
Received for publication November 5, 1999. Accepted for publication August 29, 2000.
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), primary challenged (8 days postinfection, 
),
or rechallenged (4 days postsecondary challenge, 
) mice were stained
with the indicated V
), or
secondary effector (
) specific cells were similar to those
obtained in functional assays using synthetic viral peptides measuring
intracellular IFN-
