By
From the Department of Immunology, Duke University Medical Center, Durham, North Carolina 27710
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Abstract |
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Antigen (Ag)-driven selection of helper T cells (Th) in normal animals has been difficult to
study and remains poorly understood. Using the major histocompatibility complex class II-
restricted murine response to pigeon cytochrome c (PCC), we provide evidence for both preimmune and Ag-driven selection in the evolution of Ag-specific immunity in vivo. Before
antigenic challenge, most V11+V
3+ Th (70%) express a critical complementarity-determining region 3 (CDR3) residue (glutamic acid at TCR-
93) associated with PCC peptide contact. Over the first 5 d of the primary response, PCC-responsive V
11+V
3+ Th expressing
eight preferred CDR3 features are rapidly selected in vivo. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with T cell receptor (TCR) of the
"best fit." Ag-driven selection is complete before significant emergence of the germinal center
reaction. These data argue that thymic selection shapes TCR-
V region bias in the preimmune repertoire; however, Ag itself and the nongerminal center microenvironment drive the
selective expansion of clones with preferred TCR that dominate the response to Ag in vivo.
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Introduction |
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Tcell recognition of peptide-MHC complexes is central to both the development of the preimmune repertoire and an adaptive immune response to foreign Ag. T cell development within the thymus involves ordered somatic rearrangement of gene elements for the TCR (1), followed by positive and negative selection of immature T cells based on TCR specificity (2, 3). These early selection events depend on TCR recognition of self-peptide-MHC complexes and serve to imprint the appropriate MHC restriction pattern on the preimmune T cell compartment (4, 5). After infection, preimmune T cells that recognize foreign peptide- MHC complexes are selected to participate in the immune response. This Ag-driven selection leads to T cell proliferation, effector cell differentiation, and the establishment of Ag-specific T cell memory (6). The molecular basis for these peripheral selection events and how they differ from thymic selection remains unclear.
The murine response to pigeon cytochrome c (PCC)1
has been extensively used to study TCR peptide-MHC
recognition (9). We and others have demonstrated that the
majority of PCC-specific helper T cells express V11 and
V
3 variable regions in their TCR, along with highly restricted sequences in the third hypervariable region (CDR3)
(10). Jorgensen et al. (14) established critical peptide
contact residues in the CDR3 loops of PCC-specific TCR (glutamic acid at
93 and asparagine at
100) that involved
reciprocal charge interactions with the antigenic peptide
(14). In addition, two amino acids (aa) COOH-terminal to
these contact residues, serine/threonine at
95 and alanine/glycine at
102, also appear to be preferred in PCC-specific TCR (10, 11, 13). CDR3 length appears restricted
in both chains of the PCC-specific TCR (8 aa for the
TCR-
and 9 aa for the TCR-
), with preferred J region usage that is more apparent in the TCR-
chain (J
1.2 and
2.5) than in the TCR-
chain (13). Together, these TCR
features help to describe the dominant PCC-specific clonotype and provide a means for estimating TCR diversity in vivo.
The basis for clonal dominance in an adaptive immune response remains unknown. In the thymus, a diverse preimmune repertoire can be established using a single peptide-
MHC complex (15). However, the central expression of
a foreign peptide will often deplete the preimmune repertoire of the dominant clonotype response to that peptide
(17). In an adaptive response to foreign Ag, the presence of
specific Th with restricted TCR structures provides strong
evidence for Ag-driven selection. Our earlier studies of the
PCC response indicate a high degree of restriction in the TCR of primary responders to PCC (on day 6) (13). 70%
of the primary responders already had many of the CDR3
motifs associated with PCC specificity, but the frequency
increased to 95% in the memory response (on day 6), suggesting further narrowing of the repertoire. It was not clear
when or how this repertoire narrowing occurred. Zheng et
al. indicate a rapid and progressive selection in the splenic
response to PCC that is largely complete by day 12 after Ag
priming (18). Similar studies in the class I-restricted T cell
response to allopeptides indicate no change in CDR3 diversity of the TCR- repertoire between the peak of the
primary response and the memory response (19). Similarly,
using tetramers of peptide-class I MHC, Busch and colleagues demonstrate a coordinate expansion of peptide-specific T cells (20) without particular restriction in TCR-
chain
usage (21). These workers suggest a further narrowing of
the TCR repertoire upon secondary stimulation (22), which
is not seen in other class I-restricted responses (21, 23). Although these studies are not necessarily contradictory, they
highlight the need for more comprehensive molecular
analysis of the early developing phase of the immune response.
Ag-specific Th are difficult to visualize in normal animals. Jenkins and colleagues adoptively transferred TCR-transgenic Th of known specificity into normal recipients
to monitor the dynamics of the specific T cell response (24,
25) and, more recently, cognate T-B cell interactions (26).
Their studies document the transition of Ag-specific T cells
from clusters associated with dendritic cells in the T cell
zones (27) to the B cell areas of secondary lymphoid organs
(26). In conventional animals, the V11V
3-expressing T
cells also initially expand in the T cell zones of a splenic response to PCC (18, 28). The later phase of the PCC-specific response is characterized by the germinal center (GC)
localization of V
11V
3-expressing T cells (18, 28). In
repertoire studies, Zheng et al. (18) demonstrated that the
V
11V
3-expressing cells in GC have more restricted
CDR3 loops than their T cell zone counterparts. Furthermore, the GC Th were highly sensitive to TCR- and steroid-induced apoptosis in vivo (18).
In this study, we assessed changes in TCR diversity during the emergent phase of the primary and memory response to PCC in nontransgenic animals. We directly analyzed CDR3 sequences from single, PCC-specific Th
(CD8B220
CD11b
V
11+V
3+CD44hiCD62Llo) at various timepoints throughout the immune response and
compared them to the preimmune repertoire. The memory response emerged highly restricted (days 2, 3, and 4),
with eight distinguishing CDR3 features in the majority of
PCC-specific cells. Only one of these features, a glutamic
acid at the V-J border (
93), appeared in the preimmune
repertoire at high frequencies (70% of V
11V
3-expressing Th). By day 3 of the primary response, 40% of PCC-specific cells expressed a restricted TCR containing at least
six of the eight preferred CDR3 features. Ag-driven selection in the primary response was complete by day 5, when
80% of the PCC-specific compartments expressed restricted TCR. The frequency of PCC-specific T cells with
restricted receptors remains ~80% to the end of the second week after Ag priming but increased to 96% in the memory
response. In situ analysis indicated that GC are only beginning to form on day 5, with very few PCC-specific Th
present in the GC at this stage. Overall, these data suggest
that the V
11 bias that dominates the PCC response is
centrally imposed before antigenic challenge. After initial
priming, Ag and the non-GC microenvironment selectively expand V
11V
3-expressing Th with preferred
CDR3 features that dominate the primary immune response and establish Ag-specific Th memory.
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Materials and Methods |
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Mice and Immunization
6-10-wk-old, male B10.BR mice were purchased as specific pathogen free from The Jackson Laboratory and housed under reverse barrier conditions at the Duke University Vivarium until they were killed. Whole PCC (Sigma Chemical Co.) was diluted into PBS and mixed with the Ribi adjuvant system (Ribi Immunochem Research). Primary immunization of 400 µg of PCC was injected into 200 µl of adjuvant emulsion in two 100-µl doses subcutaneously on either side of the base of the mouse tail. PBS alone was used for the adjuvant-only controls. The memory challenge was designed as a second primary immunization to reduce the differences between the two responses (i.e., no dose differences to account for changed kinetics of the cellular response). Secondary challenge was a repeat of the primary regime including adjuvant, also at the base of the tail, 8 wk after the initial priming.
Flow Cytometry
Animals were killed on various days after immunization as indicated, and the draining LNs were harvested for analysis. Inguinal and periaortic nodes were collected and teased through
80-µm mesh screens into single-cell suspensions in 0.17 M
NH4Cl solution for erythrocyte lysis before estimation of cell
count using a hemocytometer. Cells were pelleted and resuspended in PBS with 5% FCS. All cells were stained for flow cytometry at 2.0 × 108 cells/ml with predetermined optimal concentrations of fluorophore (or biotin)-labeled mAb (FITC-RR8.1
[anti-V11; PharMingen], allophycocyanin-KJ25 [anti-V
3],
PE-Mel14 [anti-CD62L; PharMingen], Cy5PE-6B2 [anti-B220; PharMingen], Cy5PE-53-6.7 [anti-CD8; PharMingen], Cy5PE-
M1/70.15 [anti-CD11b; Caltag Labs.], or biotin-IM7 [anti-CD44; PharMingen]) together with desired volume of cells on ice
for 45 min. After being washed twice, cells were resuspended at
the same cell concentration with avidin-Texas Red (TR; PharMingen) on ice for 15 min, washed again, and resuspended in
2 µg/ml propidium iodide (PI) (for dead cell exclusion) in PBS
with 5% FCS for analysis.
Samples were analyzed using a dual laser, modified FACStarPLUSTM (Becton Dickinson Immunocytometry Systems) (an argon
laser as the primary and a tunable dye laser as the secondary) capable of seven-parameter simultaneous collection (five log-amp detectors for fluorescence, one log-amp detector for obtuse light
scatter, and a photo diode for forward light scatter). The Cy5
component of the duochrome Cy5PE is also excited by the dye
laser and detected in the allophycocyanin channel. For all experiments described, the Cy5PE fluorescence collected after primary
laser excitation was used for exclusion criteria alone (see Fig. 1 A),
thereby operationally avoiding the signal overlap across the two
lasers that could not be compensated for electronically. PI was
also excluded in the Cy5PE detection channel. All analyses required the collection of two files for each sample. The first file
was a 100,000-event file of PI events to ascertain the frequency
of Cy5PE
V
11V
3-expressing cells in the total LN population. The second file contained 1,000 events of PI
Cy5PE
V
11+V
3+ cells to evaluate the fraction of cells that upregulated CD44 and downregulated CD62L. Files were acquired using
CELLQuestTM software (Becton Dickinson) and analyzed using
FlowJo software (Tree Star, Inc.). All profiles are presented as 5%
probability contours with outliers. Total cell numbers were calculated using frequencies estimated by flow cytometry and total
cell counts for the draining LNs of each animal.
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Laser Scanning Confocal Microscopy
Draining LNs used for confocal microscopy were snap frozen
in OCT embedding compound (Miles Labs., Inc.). Cryostat microtome (Leica, Inc.) cut, 6-µm-thick frozen sections were
mounted on gelatin-coated slides, air dried, and acetone-fixed for
10 min at 4°C and stored at 80°C until use. Sections were rehydrated with PBS and blocked with PBS containing 10% FCS,
10% skim milk (wt/vol) powder, and 2.4G2 (anti-FcR) (50%
vol/vol hybridoma supernatant) for 30 min at room temperature.
Sections were stained with TR-11.26 (anti-IgD), allophycocyanin-KJ25 (anti-V
3), and either FITC-RR8.1 (anti-V
11) or
FITC-Mel14 (anti-CD62L) for 1 h at room temperature, then
washed and mounted in VectorShield (Vector Labs., Inc.). FITC
is excited at 488 nm and collected using a 515-545-band pass filter, TR is excited at 568 nm and collected using a 615-645-band pass filter, and allophycocyanin is excited at 633 nm and collected using a 670-810-band pass filter. Data was acquired on a Zeiss Axiovert LSM 410 microscope system (Carl Zeiss, Inc.), and each image was collected serially in the first detector, using LSM 3.95 software. Quantitation was achieved manually after digital rendering of the image for optimal signal-to-noise and overlay using
Adobe Photoshop (Adobe Systems, Inc.). Single V
11+ and
V
3+ cells were first counted separately (and then overlapping),
using a grid covering each field (acquired using the 40× objective lens), covering one full section of the LN. The IgD staining
was then used to assign each cell's location
T zone (mainly
IgD
), B zone (mainly IgD+), or GC (IgD
area within the B
cell zone)
as well as exclude nonspecific staining (positive for all
three signals). Sections containing both T zone and B zone areas
were used for analysis.
Single-Cell Repertoire Analysis
cDNA Synthesis.
Single cells with appropriate surface phenotype were sorted for repertoire analysis using the automatic cell dispensing unit attached to the FACStarPLUSTM and Clone-CytTM software (Becton Dickinson). Each cell was sorted into an oligo d(T)-primed, 5-µl cDNA reaction mixture (4 U/ml murine leukemia virus-RT [GIBCO BRL] with recommended 1× RT buffer, 0.5 nM spermidine [Sigma Chemical Co.], 100 µg/ml BSA [Boehringer Mannheim], 10 ng/ml oligo d(T) [Becton Dickinson], 200 µM each dNTP [Boehringer Mannheim], 1 mM dithiothreitol [Promega Corp.], 220 U/ml RNAsin [Promega Corp.], 100 µg/ml Escherichia coli tRNA [Boehringer Mannheim], and 1% Triton X-100) set up in low profile, 72-well microtiter trays (Robbins Scientific), immediately held at 37°C for 90 min, and then stored atNested PCR: First Rounds of PCR (PCR-a).
2 ml of cDNA from single-cell cDNA reactions were used for two separate, 25-µl amplification reactions, one for the TCRVSecond Rounds of PCR (PCR-b).
1 µl of the first PCR product was used for further 25-µl amplification reactions for each chain of the TCR, using primers nested medially to the primers used in PCR-a (2 U/ml Taq polymerase with the recommended 1× reaction buffer [Promega Corp.], 0.1 mM of each dNTP (Boehringer Mannheim), and for [a], TCRV
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DNA Sequencing.
5 µl of PCR-b product was run on a 1.5% agarose gel to screen for positives (single bands of the right size). PCR product was then separated from primers using a CL-6B Sepharose column (Pharmacia LKB Biotechnology, Inc.). The PCR product was then directly sequenced (3 µl of PCR product, 4 µl Dye Terminator Ready Reaction Mix [Perkin Elmer Corp.], 1.5 pmol primer [V ![]() |
Results |
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To purify the PCC-specific subset, we isolated V11V
3-expressing CD4+ cells that modulate surface CD44 and
CD62L (L-selectin) expression in response to Ag. Fig. 1 A
outlines our flow cytometric strategy for purifying PCC-specific cells, using seven cellular parameters simultaneously.
This new strategy significantly decreases background to
allow more confident cell sorting, even at extremely low
target cell frequencies (<1/104 cells on day 3). The initial
background of V
11V
3-expressing cells that are also
CD44highCD62Llow is negligible before immunization (Fig.
1 A, panel iv). In the absence of protein Ag, not only on
day 3 but also through to day 7, there is negligible appearance of Ag-responsive cells (Fig. 1 B, top row). There is a
significant difference between adjuvant-only versus day 3 PCC-responsive cells (P = 5.0 × 10
4) (Fig. 1 C). In addition, the response to an irrelevant protein, such as hen egg
lysozyme, is similarly low (data not shown). These critical
in vivo controls attest to the specificity of cells responding to PCC.
The increase in frequency for Ag-responsive V11V
3
cells (CD44hiCD62Llo) is depicted in the probability contours of Fig. 1 B. It is important to note that the total cellularity of the draining LNs also changes over the course of
the immune response. Therefore, it is more informative to
consider the change in total cell numbers of Ag-responsive cells over the course of the response (Fig. 1 C). We observed a 250-fold increase in cell numbers between days 0 and 7 of the primary response. Of course, the fidelity of the
day 0 quantitation is limited by detection and not the actual
precursor frequency in the preimmune repertoire. There is
an apparent plateau in cell numbers from days 7 to 9 of the
primary response and then a gradual decline. The extent of
the cellular response to the secondary challenge with the
same dose of Ag is very similar to the primary response. It is
the accelerated kinetics of this cellular response that highlights one of the unique characteristics of a memory Th response (peak on day 3; Fig. 1, B and C) (13).
Using the
flow cytometric strategy outlined above, we can isolate single PCC-responsive cells from the emergent phase of both the primary and memory response to Ag. We first focus
our attention on the memory response. We had previously
defined the expression of highly restricted TCR on day 6 of the memory PCC response (13). It was not known
whether the restricted TCR expressed on day 6 were the
result of clonal maturation following secondary challenge. Therefore, we isolated single cells from days 2, 3, and 4 for repertoire analysis, as described in detail in Materials and
Methods. Regardless of the day after challenge, TCR expressed by memory response cells are highly restricted
(sequences from days 2 and 4 are displayed in Fig. 2). The
similarity between aa sequences from different cells can be
easily seen; however, clonal relatedness can only be established by comparing DNA sequences of both TCR- and
TCR-
chains from single cells. Identical sequences for
both chains were seen in only 6/46 TCR sequences from
the memory response (across four separate animals). These
repeat sequences were not amplification artifacts (which are
rigorously scrutinized in the experimental design) and
therefore represent examples of single cells from the same
parent clone in vivo. Overall, these data demonstrate that
the memory response to PCC emerges rapidly, using a
broad array of memory-response precursor cells that already express highly restricted TCR.
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Using this more complete data set, we can define four
preferred CDR3 features (in each chain of the TCR) that
typify the PCC-specific memory Th compartment. In the
TCR- chain (Fig. 2): (i) glutamic acid (E) at
93; (ii)
CDR3 length of 8 aa; (iii) serine (S) at
95; and (iv) J
16, 17, 22, and 34. In previous studies, a threonine was also
seen at
95, but it was not seen in this study (0/51 TCR-
chains). We can now assign J
16, 17, 22, and 34 as preferred, with each J
used by >10% of the memory
responders and together accounting for 70% of memory
responders. These four J
can produce a serine at position
95 given the appropriate V-J junction; however, the four
J
represent only a subset of total J
segments that can create this serine (at least 10 others). Therefore, the J
segments are preferred for reasons other than simply the creation of a serine at
95. The four preferred features in the
TCR-
chain are: (i) asparagine (N) at
100, (ii) CDR3
length of 9 aa, (iii) alanine (A) or glycine (G) at
102, and
(iv) J
1.2 and J
2.5. The
102 position is considered separately from the J
1.2, as most often, the alanine appears
to be lost in D-J joining in the preimmune repertoire (4/13
J
1.2 expressing preimmune TCR retain the alanine, and
only two of these express the alanine at the correct position). Therefore, J
1.2 is not the preferred motif but
rather a J
1.2 that retains an alanine at position
102. A glycine is also found in the
102 position when J
2.5 is
used. In these cases, the glycine is encoded by D region or
N sequence insertions and is not present in the germline
J
2.5.
To evaluate when the dominant clonotype emerges, we next sorted
single PCC responder cells from throughout the primary
response. The dot plot displays in Fig. 3, A-D and Fig. 4,
A-D summarize the CDR3 sequence information for either the TCR- or TCR-
chains from over 500 single
cells. Each dot represents the sequence from a single cell,
and each of the eight CDR3 features from these cells are
displayed separately. The preferred CDR3 motif is presented at the top of each panel, with alternative features
displayed in order of prevalence. A summary for each chain
in Figs. 3 E and 4 E combines all CDR3 features from individual animals to demonstrate when the repertoire narrows
over the course of the developing immune response.
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Considering all eight preferred CDR3 features, only the
glutamic acid at 93 preexists antigenic challenge to any
significant extent (Fig. 3 A). In preimmune and PCC-nonresponsive Th (V
11+V
3+CD44loCD62Lhi), 68% express
glutamic acid at position
93 (n = 62). In the same population, the three other preferred TCR-
chain features are
present in <30% of the cells (Fig. 3, B-D). The glutamic
acid at
93 is encoded by the last codon of the V region
and may be lost on imprecise V-J joining. Its presence in
the preimmune repertoire may be simply stochastic or the
result of thymic selection pressures. Greater than 90% of all
V
11V
3-expressing T cells in the periphery of normal
B10.BR mice are CD4+, implicating thymic selection and
MHC class II restriction as a defining characteristic of the
preimmune repertoire for these particular T cells (data not
shown) (13). Of 75 murine TCR-
regions listed by
Arden et al. (29), only four can express a germline-encoded
glutamic acid at
93. All four V regions are V
11 subfamily members. Therefore, it seems likely that the presence of
this one critical peptide contact residue that preexists antigenic challenge at very high frequencies in the V
11V
3-expressing Th imposes the TCR-
chain V region bias of
the I-Ek-restricted PCC response.
The remaining seven preferred CDR3 features are rapidly
selected during the cellular expansion phase of the primary
response (days 3-7; doubling time of the population was
17.5 h). Even by day 3 of the primary response, there is an
accumulation of Ag-activated V11V
3 cells with many
of the preferred CDR3 features (Fig. 3, B-D and Fig. 4,
A-D). There is a large spread of J
usage in the PCC-nonresponsive cells that has already narrowed by day 3 (35%) and narrows further (65%) to use the four preferred J
s by
day 5 (Fig. 3 B). The CDR3 lengths of 8 and 9 aa are most
prevalent in the PCC-nonresponsive cells; however, the
preference for a length of 8 aa in the PCC-specific compartment is evident even by day 3 and maximal by day 5 (Fig. 3 C). Selection for serine at
95 also appears maximal
by day 5 of the primary response (Fig. 3 D).
Many PCC-specific hybridomas contain at least three
out of the four CDR3 features described. Therefore, in
Fig. 3 E, we consider the change in the frequency of cells
that express 3 preferred CDR3 features in their TCR-
chains to assess the dynamics of clonal maturation. By day 3, there is a significant difference in cells that express
3 preferred features over the PCC-nonresponsive cells (P = 0.01, 2-tail t test). There is a further increase in frequency
of restricted TCR by day 5 (days 3-5, P = 0.01) but no
significant difference over the course of the primary response. This was also true between the late primary response and the memory response. Furthermore, we found
no evidence for somatic diversification of the TCR-
genes (30) (no mutations observed for 5,441 bases analyzed
for the TCR-
chain from days 7, 9, and 11; n = 83 single cells, 40-90 bp upstream of the CDR3 in each case).
A similar rapid progression of Ag-driven selection was
apparent for the TCR- chain. Very few PCC-nonresponsive cells express an asparagine at
100 (6%), with
evidence for selection in the PCC-specific compartment by
day 3 (20%) and clearly by day 5 (65%) (Fig. 4 A). This
preferred CDR3 feature appears further selected by day 7 (73% by day 7; 78% average over days 7-14), with still further selection in the memory response (90% average over
days 2-4). Preferred J
usage (1.2 and 2.5) follows a similar
course (Fig. 4 B): a small increase in J
1.2 and 2.5 usage on
day 3 (23% resting to 34% by day 3) that is clearly dominant by day 5 (77%), with a further increase by day 7 (88%;
88% average, days 7-14) and a slight increase in the memory response (94% average). CDR3 length restriction may
be more rapid than the previous two features (Fig. 4 C).
The appearance of PCC-specific cells with a 9-aa length is
close to maximal frequencies by day 5 (87%), with little
further change in the primary (93% average days 7-14) and
memory (96% average) responses. Appearance of alanine or
glycine at
102 follows the kinetics of the first two TCR-
chain features presented (Fig. 4 D). There is a small increase in prevalence noticeable by day 3 (33% resting to
46% by day 3) that is clearly dominant by day 5 (80%), with
a further increase by day 7 (96%; 94% average, days 7-14)
and little change in the memory response (97%).
The summary in Fig. 4 E presents the change in frequencies of cells that express 3 preferred CDR3 features across
multiple animals. There is a significant increase between
the resting cells and PCC-specific cells by day 3 (P = 0.04, 2-tail t test) and the greatest change between days 3 and 5 (P = 0.01). Although there appear to be some differences
between days 5 and 7 for the individual CDR3 features
discussed above, when considered together in this summary, there was no statistically significant difference. There
is also no apparent difference between day 7 and week 2 of
the response nor any difference between these days and the memory response. Therefore, we conclude that a rapid
maturation in the PCC-specific Th compartment for
clones that express these preferred TCR-
CDR3 features
is largely complete by day 5 of the primary response. We
found no evidence for somatic diversification of the TCR-
chain (31) (no mutations observed for 3,406 bp analyzed
for the TCR-
chain from days 7, 9, and 11; n = 104 single cells, 20-40 bp upstream of the CDR3 in each case).
In Fig. 5, we summarize sequence information
from both chains of the TCR of single, V11V
3-expressing Th (n = 245; a subset of cells from Figs. 3 and 4). In
the dot plot display, we emphasize the emergence of PCC-specific cells that express
6 of the preferred CDR3 features described above as the change in their frequency over
time. Only 1/47 resting V
11V
3 cells expressed
6 preferred CDR3 features. By day 3, 40% of the PCC-responsive compartment (V
11V
3CD44hiCD62Llo; n = 50) already expressed
6 preferred CDR3 features. By days 5-7,
this frequency doubled to 83% (n = 59; these days have
been combined to present similar numbers in each group.
There was no significant difference in frequency of cells
with
6 preferred features between these two timepoints).
There was no further change in frequency of these restricted TCR to day 14 of the primary response (n = 43).
After secondary challenge, 96% of PCC responders expressed
6 preferred CDR3 features (n = 46). This further
increase in restricted responders may indicate a separate
phase of Ag-driven selection associated with the induction
of a memory response. Overall, these data consider the
complete TCR as the selecting unit and further attest to
the rapidity of Ag-driven selection in this system.
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Fig. 6 displays
a representative set of TCR sequences from PCC-responsive, V11V
3-expressing Th from day 3 of the primary response. These sequences can be divided into three groups
based on the degree of restriction in their CDR3 regions.
40% of day 3 responders expressed
6 CDR3 features associated with the PCC response (Fig. 6, Group 1). The
second group is designated as unrestricted (containing
5
preferred CDR3 features), but expressed TCR-
chains
similar to those sequenced from PCC-specific hybridomas, PCC-specific cell lines, or binders of moth cytochrome c
(MCC)/I-Ek tetramers (Fig. 6, Group 2). Clones, such as
the well-characterized 2B4, fall into this category, with a
TCR-
chain that expresses none of the preferred CDR3
features we have found in the memory PCC response but
is clearly specific for PCC. These first two groups account
for 80% of the cells from day 3. Cells in the third group
make up the remaining 20% of PCC responders from day 3 and expressed unrestricted TCR (
5 preferred CDR3 features) that have not been previously associated with PCC
specificity (Fig. 6, Group 3). It is important to note that not
only was the cellular response on day 3 significantly above
the adjuvant-only control (the main in vivo criteria for
specificity), but the few events that were sorted from adjuvant-only controls (n = 60) gave rise to a PCR product
with a sixfold lower efficiency (see Materials and Methods for details). The few TCR sequenced from these control
populations were as diverse in their CDR3 as their preimmune counterparts (data not shown). Overall, these data
indicate that PCC responders initially recruited into the
immune response express more diverse TCR. Subsequently, the Th with preferred TCR are selectively expanded, and a subset of these cells are preserved for the
memory response.
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We have determined that Ag-driven selection in the draining LNs is largely complete by day 5 of the primary response. In Fig. 7, we outline a quantitative analysis of the
GC and non-GC distribution of PCC-specific Th over the
course of the primary response. It should be noted that
>90% of V11V
3-expressing T cells in the LN of
B10.BR mice are CD4+ Th (by flow cytometric analysis;
data not shown). In Fig. 7 A, we display an example of
three-color laser scanning confocal microscopic (LSCM)
imaging to localize V
11V
3-expressing T cells (yellow). IgD staining is used primarily to delineate B cell zone
(IgD+) and T cell zone (IgD
) but also to locate the IgD
regions within the B cell zones that indicate the presence of GC. In Fig. 7 B, we compare our quantitation of V
11-
and/or V
3-expressing T cells by LSCM analysis and flow
cytometry. Quantitation was undertaken from either
100,000 event flow cytometric files or cell counts from
entire cross-sections of LN tissue. The concordance for
proportions of the single-positive (V
11 or V
3) and double-positive (V
11V
3) cells within the LN populations
between LSCM analysis and flow cytometry is high (Fig. 7
B). Reproducibility is also high across different animals (as
indicated by the SEM; Fig. 7 B). In Fig. 7 C, the GC and
non-GC distribution of V
11V
3-expressing T cells across
days 5, 7, and 9 is presented, and the unadjusted graphical
representation of this data is shown in Fig. 8 A. From these
data, it is clear that the GC reaction is in its very early stages
on day 5 of the LN response, increasing by day 7 and increasing further on day 9.
|
|
Not all V11V
3-expressing T cells are PCC specific.
Our flow cytometric analysis has focused on CD62L
downregulation as one index for activation within the
V
11V
3-expressing compartment, and we can calculate
the fraction of the total V
11V
3 compartment that is
CD62Llo at any stage of the response in vivo. Even at the
peak of the primary response, only about half of the
V
11V
3-expressing cells in the draining LNs are PCC
specific (by flow cytometry and TCR sequence analysis;
Fig. 1). Using LSCM analysis, we demonstrate that all
V
3+ cells in the GC (the majority of which are V
11+;
data not shown) are also CD62Llo (Fig. 7 D). Combining
the flow cytometric and LSCM data, we can calculate the
proportion of V
11V
3 cells in the non-GC compartment that are not PCC specific. Fig. 8 B presents the adjusted
distributions for PCC-specific cells over the course of the
primary response and highlights the coincident decline in
the non-GC compartment with the increase in the GC
compartment. In Fig. 8 C, we present the expansion and
decline of total PCC-specific cells (from flow cytometric
data in Fig. 1 C, on a linear scale to emphasize the decline
phase of the response) and then apply the frequencies of
GC and non-GC V
11V
3 T cells calculated by the
LSCM analysis to illustrate the emergence and decline of
the total PCC-specific compartment in these distinct microenvironments. We find that the plateau phase of the cellular response demonstrated by flow cytometric analysis
(between days 7 and 9 of the primary response; Fig. 8 C)
resolves into two peaks when the microenvironment is taken into account (Fig. 8 D). The first peak indicates maximal non-GC cell expansion (day 7), and the second peak
indicates GC cell expansion (day 9). Whether these two
peaks are the result of migration alone or migration and
then proliferation is not clear from our data. Nevertheless,
with Ag-driven selection virtually complete by day 5 of the
primary response, the more delayed kinetics of the GC T
cell pathway strongly argue that Ag-driven selection is a
non-GC activity.
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Discussion |
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Our study documents the evolution of clonal dominance
in vivo. We believe that these processes are fundamental to
the development of highly specific Th-based regulation of
primary immune responses. The PCC model allows experimental access to a Th response that becomes dominated by
Ag-specific Th expressing highly restricted TCR. The
dominant PCC-specific cells exhibit a bias in V region usage (V11V
3) and TCR with preferred CDR3 features
that provide molecular indicators of TCR diversity. In this
study, we demonstrate that 70% of all V
11+V
3+ Th express a critical PCC peptide contact residue (glutamic acid at
93), even before initial antigenic challenge. However,
PCC-specific cells with all eight CDR3 features associated
with the dominant clonotype only emerge to detectable
levels after initial priming with PCC. Although there is
some increase in the frequency of the dominant PCC-specific clonotype between the primary and memory responses
(81-96%), the majority of Ag-driven selection occurs very
rapidly during the first 5 d after initial priming. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with the "best fit" TCR.
The TCR repertoire narrows before significant GC expansion, implicating Ag and the non-GC microenvironment as
the principle selecting influences in vivo.
Of all eight
preferred CDR3 features used by the dominant clonotype,
only the glutamic acid at 93 of V
11 preexists antigenic challenge to any significant degree. The prevalence of this
residue is likely to impose the V
11 dominance associated
with PCC specificity in I-Ek-restricted animals. In PCC-specific hybridomas from many sources, V
11 is more
consistently expressed than V
3 (10, 11, 17, 32, 33). In
studies of single chain TCR-transgenic animals, immunization with analogue peptides of MCC altered the V region dominance of PCC-specific responders (14). The V
3
dominance was more readily perturbed than V
11, presumably due to modification of Ag-driven selection. Manipulations of the thymic selecting environment can also perturb V region dominance in the response to PCC. When
wild-type or analogue peptides of MCC are introduced centrally, the V
3 dominance of MCC responders in the
periphery is more noticeably affected than V
11 (17, 32).
These central manipulations are most likely to alter the
availability of particular clonotypes in the preimmune repertoire rather than directly affect Ag-driven selection.
The presence of glutamic acid at 93 is not the only feature that predisposes V
11V
3-expressing Th in the preimmune repertoire to bind PCC epitopes. Most V
11V
3
Th on days 3 and 5 after initial priming that have not modulated CD44 or CD62L also express glutamic acid at
93.
Therefore, the combination of V
11 with the glutamic acid at
93 and any V
3 V region are not sufficient for
PCC specificity. Furthermore, V
11-expressing Th after
PCC immunization that do not bind tetramers of MCC-
I-Ek also retain a predominance of glutamic acid at
93
(67% of D8 and D14; n = 12) (McHeyzer-Williams, L.J.
and M.G. McHeyzer-Williams, unpublished data). Therefore, the glutamic acid at
93 may impose the V
11 bias
seen in PCC responders, but other particular TCR features
are also clearly required for fine specificity. These other
features are not easily recognized in the TCR of PCC responders initially recruited in the response (isolated on day
3 of the primary). It is possible that the early responders
represent a stochastic selection from the V
11V
3 Th subset of preimmune Th from which the dominant clonotype
is then selected. In this latter scenario, subsequent Ag-driven
selection events only focus on the cells initially recruited.
This would explain why there is no obvious depletion of
V
11V
3 Th from the nonresponder population. To argue against this stochastic model, only a minute fraction of
all preimmune V
11V
3 Th (0.04%) are able to bind tetramers of MCC-I-Ek (McHeyzer-Williams, L.J., J.F. Panus, J.A. Mikszta, J.D. Altman, M.M. Davis, and M.G.
McHeyzer-Williams, manuscript in preparation). Overall,
it is more likely that the TCR structural requirements for
early recruitment into the PCC response are less stringent and, therefore, more difficult to identify.
TCR
specificity evolves rapidly after primary exposure to Ag in
vivo. By day 5 of the primary response, >80% of PCC-specific Th express restricted TCR (6 preferred CDR3
features). The biochemical basis for Ag-driven selection in
vivo is still not clear. Lanzavecchia and colleagues demonstrate the utility of having TCR with high off-rates to enable serial triggering of multiple receptors (34, 35). In this
model, lower affinity TCR receptors may be preferred and
used for memory responses. In their recent study, Crawford et al. demonstrate a hierarchy of affinities for a series of
PCC-specific hybridomas (using biacore analysis and correlated levels of MCC-I-Ek tetramer staining) (36). The
KMAC-92 hybridoma (6/8 preferred CDR3 features) has a
Kd of 29 µM (33), whereas the well-characterized 2B4 hybridoma (3/8 preferred CDR3 features) has a Kd of 90 µM (37, 38). Single cells with TCR similar to 2B4 can be
found early in the primary response but do not appear to be
selected into the memory compartment. Furthermore, the
AD10 and TCR-transgenic cells in the study by Crawford
et al. (36) both express eight preferred CDR3 features
(similar to the 5C.C7 TCR). Tetramers of peptide-MHC
complexes have been used for analysis of class I-restricted
responses in conventional animals (22, 23, 39) and class
II-restricted responses in single-double-chain transgenic animals and T cell hybridomas (36, 44). These tetrameric
reagents provide the means to assay affinity directly ex vivo
in both class I- and class II-restricted responses.
Ag-driven
selection of the preferred clonotypes is enhanced by selective cellular expansion in vivo. The preferred clonotype is
already present on day 3 of the primary response (40%
prevalence). Whether the presence of the day 3 PCC-specific cells already represents cell expansion or simply recruitment from distant sites is not yet clear. Nevertheless, the
day 3 PCC-specific compartment expands a further 20-fold
before reaching a plateau on day 7. Although the maximal
frequency of preferred clonotypes is reached by day 5 (80%),
there is still further expansion of this already restricted cell
population up to day 7 (Fig. 1). Our data provides a glimpse of TCR structures that are initially recruited but are not
further expanded (or preserved) in the response to PCC
(day 3 sequence data; Fig. 6). Although the TCR from
these early responders are less restricted than the dominant
clonotype, similarly diverse CDR3 structures have been
observed in the TCR- chain of many PCC-specific hybridomas (Fig. 6, Group 2) (11, 17, 32, 33). The hybridomas have been selected in vitro, with excess amounts of
specific Ag providing no selective pressure between PCC-specific clones. In contrast, there may be significant selection pressure between clones in vivo, as Ag depots recede
over the course of the response. We and others have documented similar diversity in the TCR-
of T cells that bind
tetramers of MCC-I-Ek (36) (McHeyzer-Williams, L.J.,
J.F. Panus, J.A. Mikszta, J.D. Altman, M.M. Davis, and
M.G. McHeyzer-Williams, manuscript in preparation).
We see no downregulation of TCR during the PCC-specific response, as occurs in vitro after Ag stimulation
(45). Although our isolation strategy clearly relies on TCR
expression, there is no difference in levels of TCR between
the PCC-specific cells and the nonresponder V11V
3-expressing population (data not shown). These data further
argue that Ag may be limiting in vivo (at least by day 3 after initial priming). It is also possible that some V
11V
3-expressing, PCC-specific clones recognize different peptide epitopes. The failure of particular clones to expand may
correspond to the relative lack of availability of different
epitopes over the course of the response, as suggested by
Butz and Bevan for class I-restricted responses (46). It
would be surprising if TCR specific for completely different epitopes used the same V region pair with similar
CDR3 features. It is more likely that there may be subtle
differences in the nature of the selecting peptide early in
the response due to Ag processing or APC type (with different costimulatory molecules).
Our data favor a
simple model of preferential clonal expansion that conforms to the edicts of the clonal selection theory (47). In
this model, there is an initial recruitment of cells expressing
the appropriate V region genes with particular bias toward
V11 usage as well as V
3. This initial set of PCC-specific cells has more diverse TCR than the dominant clonotype
but is more restricted in its CDR3 loops than in the preimmune compartment. The clones expressing all preferred
TCR structures are then selectively expanded from this initial pool and dominate rapidly through cellular expansion.
Both the rapid kinetics of Ag-driven selection and the
highly restricted memory response suggest that focusing of TCR specificity precedes memory cell development. There
is a further increase in the frequency of restricted TCR in
memory responders over the late primary responders (81-
96%) that could suggest another phase of selective expansion after secondary challenge. The highly restricted TCR
of memory cells may underpin the rapid cellular expansion
that typifies the response to secondary antigenic challenge.
There were suggestions of clonal maturation in the Th compartment in
our earlier study of the PCC response (13). The initial study suffered from two technical limitations that have
been overcome in the current analysis. The first involves an
emphasis on population analysis. The majority of the CDR3
sequence analysis was presented for the V3 chain only,
from populations of 1,000 cells as the starting point for
RT-PCR. Whereas the same trends were apparent in the
limited single-cell survey presented at that time (n = 12 from each of the primary and memory response), single-cell resolution of this study was required to provide confidence in the changes in frequency of the dominant clonotype over time (combining both cellular and molecular
analyses). The second technical difficulty was the very low
frequency of PCC-specific cells at the early stages of the
primary response. The addition of the seventh parameter in
the flow cytometric analysis reduced the background at
least 10-fold. The use of an exclusion channel (excludes not
only cells outside the lineage of interest but also cells that
nonspecifically bind antibodies). In addition, the use of CD44
and CD62L, together with the TCR-specific reagents,
greatly clarified the day 3 and 5 selection of PCC-specific
cells. With this new strategy, we extended our initial survey (two timepoints, day 6 of the primary and day 6 of the
memory) to the extended timecourse needed to resolve the
dynamics of clonal selection in vivo.
The emergence of PCC-specific Th in the non-GC and GC microenvironments of the draining LNs of these animals is in general agreement with early studies of the splenic T cell response to this Ag (18, 28). Our analyses of repertoire narrowing are similar to the studies of Zheng et al. (18); however, the rate and extent of selection in our current study appears far more rapid. The apparent slower rate may be due to differences between the splenic and LN microenvironments that regulated these processes. Alternatively, differences may be due to the phenotypic selection used for repertoire studies in each case. The splenic PCC response also appears more restricted in the GC environment than non-GC at the same timepoint of analysis. Zheng et al. imply that Ag-driven selection is occurring in the GC and demonstrate that GC T cells are highly susceptible to CD3-mediated apoptosis resembling thymic development and selection (18). In the LN, the vast majority of Ag-driven selection is over before significant expansion of the GC compartment.
It appears unlikely that the GC reaction plays a role in the repertoire narrowing itself; it rather appears to be a site for migration of already restricted PCC-specific Th. The Ag-specific GC Th continue expanding in vivo (18, 28) and differentiate into effector cells that support the development of B cell memory (48, 49). Furthermore, we see no evidence for the somatic diversification of either chain of the TCR in this study, as previously reported (30, 31). This was also true on day 9 of the primary response, when 75% of the PCC-specific compartment resides in the GC (Fig. 8). Given the kinetics of cellular expansion in the LNs, early T zone proliferation associated with APC-Th conjugates is the most likely location for the selective expansion of preferred clonotypes (25, 27).
Conclusions.TCR specificity evolves rapidly through the preferential expansion of Ag-specific T cells well before the peak of the initial cellular response to Ag priming. These earliest events help to regulate the nature of effector cell function and shape the final specificity of the long-lived memory compartment. Here, we demonstrate not only the TCR structures of the preferred clonotypes and the kinetics of their selection, but also the TCR structures of clones initially recruited into the specific response but not expanded significantly for effector function or preserved into the memory compartment. These studies provide the framework for understanding the biochemical basis and functional consequences of maturation in the Th compartment.
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Footnotes |
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Address correspondence to Michael G. McHeyzer-Williams, Duke University Medical Center, Department of Immunology, Rm. 316 Jones Bldg., Research Dr., Durham, NC 27710. Phone: 919-613-7821; Fax: 919-684-8982; E-mail: mchey002{at}acpub.duke.edu
Received for publication 31 December 1998 and in revised form 19 March 1999.
J.A. Mikszta was a recipient of a National Research Service Award fellowship. This work was supported by an Arthritis Foundation Biomedical Sciences Grant and National Institutes of Health grant AI40215.We would like to thank Rebecca Caley, Gabriel Bikah, David Driver, and Garnett Kelsoe for constructive comments and critical review of the manuscript. We also thank Maria Karvelas for expert technical advice regarding confocal microscopy and the Duke Comprehensive Cancer Center Confocal Microscopy Facility. Special thanks to J. Michael Cook and the Duke Comprehensive Cancer Center Flow Cytometry Shared Resource.
Abbreviations used in this paper aa, amino acids; CDR3, complementarity-determining region 3; GC, germinal center; LSCM, laser scanning confocal microscopic; MCC, moth cytochrome c; PCC, pigeon cytochrome c; PI, propidium iodide; RT, reverse transcriptase; TR, Texas Red.
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