By
From the Department of Pathology and Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
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Abstract |
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Numerous studies have examined T cell receptor (TCR) usage of selected virus-specific T cell
clones, yet little information is available regarding the stability and diversity of TCR repertoire usage during viral infections. Here, we analyzed the V8.1 TCR repertoire directly ex vivo by
complementarity-determining region 3 (CDR3) length spectratyping throughout the acute
lymphocytic choriomeningitis virus (LCMV) infection, into memory, and under conditions of
T cell clonal exhaustion. The V
8 population represented 30-35% of the LCMV-induced
CD8+ T cells and included T cells recognizing several LCMV-encoded peptides, allowing for
a comprehensive study of a multiclonal T cell response against a complex antigen. Genetically
identical mice generated remarkably different T cell responses, as reflected by different spectratypes and different TCR sequences in same sized spectratype bands; however, a conserved
CDR3 motif was found within some same sized bands. This indicated that meaningful studies on the evolution of the T cell repertoire required longitudinal studies within individual mice.
Such longitudinal studies with peripheral blood lymphocyte samples showed that (a) the virus-induced T cell repertoire changes little during the apoptosis period after clearance of the viral
antigens; (b) the LCMV infection dramatically skews the host T cell repertoire in the memory
state; and (c) continuous selection of the T cell repertoire occurs under conditions of persistent
infections.
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Introduction |
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Viral infections often induce potent immune responses associated with hyperplasia of T cells, which clear the virus, undergo apoptosis, and return to homeostasis, leaving the host with memory T cells that rapidly respond to the pathogen on subsequent challenge (1). Although many studies have examined the specificities and functions of selected virus-specific T cell clones (2), little is known about the diversity of the T cell repertoire in genetically identical hosts responding to infection and how this repertoire changes during the transition between an acute infection and a memory response. The relationship between T cell repertoires during primary and secondary viral infections and the evolution of the T cell response under conditions of clonal exhaustion associated with persistent infections are also not well understood. For instance, Are there changes in the T cell repertoire during the apoptotic phase of the T cell response? How similar is the memory repertoire to that of the acute infection? Does T cell clonal exhaustion under conditions of high antigen dose eliminate all or simply a subpopulation of responding T cells?
The strong T cell response during lymphocytic choriomeningitis virus (LCMV)1, strain Armstrong (LCMV-ARM) infection of C57BL/6 mice is well characterized and provides a useful system to study the evolution of the TCR repertoire under conditions where antigen is cleared and where an acute T cell response converts into a memory response. The clone 13 variant of the Armstrong strain, which differs from its parent strain by only two amino acids, disseminates broadly in vivo and results in persistent infection and clonal exhaustion of CTL in high doses (6, 7). The CD8+ T cell epitopes of LCMV-ARM and LCMV clone 13 are the same (8), thereby enabling one to compare the evolution of the LCMV-induced TCR repertoire usage during the LCMV-ARM and LCMV clone 13 infections. For the rest of this paper, we use LCMV to represent the LCMV-ARM parent strain, and clone 13 to represent the disseminating variant.
The acute LCMV infection causes a logarithmic expansion of LCMV-specific CTL, peaking at ~1/30 precursor CTL (pCTL) per CD8+ cell at days 8-9 after infection (9). These activated T cells become sensitized to activation-induced cell death and die through apoptosis at days 10-14 after infection (10, 11). Although there is a remarkable reduction in the total number of CD8+ cells, the pCTL frequency per CD8+ cell for each of three immunodominant peptides drops only twofold and thereafter remains stable throughout the lifetime of the mouse (9, 12). The LCMV-specific CD4+ T helper cell precursors (pTh) peak at only about 1/600 CD4+ T cells, but reduction of pTh per CD4+ cell and the stability of LCMV-specific pTh are similar to those observed in the CD8 system (1). In contrast, the CTL memory is lost under conditions of high doses of LCMV clone 13 infection, which induces a transient antiviral CTL response followed by a clonal exhaustion of the virus-specific CTL (7).
Kinetic studies on the pCTL frequencies to three immunodominant peptides have suggested that the LCMV-specific T cell repertoire remains unchanged between day 7 of infection and well into long term memory and that a high frequency of LCMV-specific memory T cells is preserved into memory. However, these data are dependent on the ability of T cells to grow out in limiting dilution assays (LDA), where in vitro manipulation in the functional assays might introduce unknown biases. Recent work using tetrameric MHC molecules to quantify LCMV-specific T cells supports our previous study, which concluded that most of the CD8+ T cells included during LCMV infection are virus specific (13, 14). These studies also support our LDA data that show a substantial skewing of the TCR repertoire toward virus-specific cells in the memory state. However, these studies define T cells in terms of their specificity and do not evaluate any changes in TCR usage that may develop during the progression of an infection. Little is known about the relationship of multiple clones of T cells to each other in a complex multiclonal immune response directed against a number of immunodominant and subdominant peptides.
To evaluate the dynamics of such a multiclonal T cell response to a virus infection we have done a molecular analysis of the TCR repertoire directly ex vivo by complementarity-determining region 3 (CDR3) length spectratyping,
a technique initially developed by Pannetier et al. (15).
This technique consists of two major steps to analyze the
CDR3, which is encoded by the joining of V, D, and J
segments and defines much of the TCR specificity (16). First, the RT-PCR reaction with specific V and C
primers competitively amplifies, from a bulk population of
T cells, TCR with specific V
sequences but different
CDR3 regions. Second, the PCR products are further analyzed with different labeled J
primers in a run-off reaction
to yield the size peaks for all the VDJ combinations. The
spectrum of size differences across the CDR3 region is
known as the spectratype. Since the relative intensity of a
given size peak is proportional to the amount of the nonamplified RNA molecules, an increase in the height and
area of a particular peak signals expansion of T cell clones.
Therefore, one can obtain distinct CDR3 profiles to analyze for changes of the TCR repertoire during infection.
To examine the LCMV-induced T cell repertoire usage,
we analyzed the V8.1+ T cell repertoire because it was
expanded vigorously during acute LCMV infection. It represents a substantial portion of the CD8+ T cell response
and includes T cells with a wide variety of specificities directed against several virus-encoded peptides. Our data indicate that genetically identical mice generate significantly
different T cell responses to the same antigens, albeit with
some preferential J
usage and a conserved TCR motif.
This indicates that, unlike LDA and tetrameric MHC-peptide analyses, spectratype comparisons of T cell receptors
between mice are uninterpretable and that individual mice
must be examined longitudinally for a meaningful analysis
of the evolution of T cell responses. We present here such
a longitudinal study, and our data support the concepts that (a) no further selection of LCMV-induced T cell repertoire
occurs after the virus is cleared and during the apoptotic
phase of the immune response; (b) the LCMV infection alters the host T cell repertoire dramatically and leaves it
with a high frequency of LCMV-specific T cells in the
memory state; and (c) evolution of the T cell response occurs under conditions of clonal exhaustion associated with
persistent infection.
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Materials and Methods |
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Virus Preparation.
The LCMV-ARM strain and the AN3739 strain of Pichinde virus (PV) were propagated in baby hamster kidney cells, as described previously (17). The highly disseminating LCMV-ARM clone 13 variant was obtained from Dr. M.B.A. Oldstone (Scripps Clinic and Research Foundation, La Jolla, CA) (8).Infection of Mice.
5-6-wk-old C57BL/6 male mice were purchased from The Jackson Laboratory (Bar Harbor, ME). For viral infection, mice were injected intraperitoneally with 4 × 104 PFU of LCMV or 3 × 106 PFU of PV in a 0.1-ml vol per mouse, or intravenously with 2 × 107 PFU of LCMV clone 13 in a 0.4-ml vol per mouse. To reduce nonspecific T cell activation induced by fetal bovine serum, LCMV and PV were diluted at 1:100 and 1:20, respectively, with phosphate-buffered saline before injection. For secondary LCMV infection, the immune mice at 6-7 wk after infection, before neutralizing antibodies were significantly induced (18), were injected intraperitoneally with 107 PFU of LCMV in a 0.2-0.3-ml vol per mouse.Spleen Cell Preparation and Sorting.
Spleen leukocytes were prepared as described previously (19). To isolate CD8+VAntibody and Complement Depletion.
To deplete CD4+ cells, spleen cells were incubated with anti-CD4 (GK1.5) for 45 min on ice, washed once with RPMI 1640, and then incubated with MAR 18.5 for 45 min on ice. The cells were washed once with RPMI 1640, treated with rabbit complement H2 (Pel-Freez Clinical Systems, Brown Deer, WI), and then incubated at 37°C in a humidified 5% CO2 incubator for 45 min. At the end of the incubation, the cells were washed three times with RPMI 1640.Target Cell Preparation.
MC57G (H-2b), a methylcholanthrene-induced fibroblast cell line from C57BL/6 mice, was propagated in MEM (GIBCO BRL, Gaithersburg, MD) supplemented with 100 U/ml of penicillin G, 100 µg/ml streptomycin sulfate, 2 mM L-glutamine, 10 mM Hepes (United States Biochemical Corp., Cleveland, OH), and 10% heat-inactivated (56°C, 30 min) fetal bovine serum (Sigma Chemical Co.). The TAP-2-deficient cell line RMA-S (H-2b) was from Hans-Gustaff Ljunggren (Karolinska Institute; Stockholm, Sweden) and was grown in RPMI 1640, supplemented as above. To prepare target cells, MC57G cells were infected with LCMV or PV at a multiplicity of infection of 0.1 PFU/cell and incubated for 2 d at 37°C. RMA-S cells were pulsed overnight with 200 µM of LCMV peptide glycoprotein 33 (GP33), GP276, or nucleoprotein 396 (NP396), as described previously (20).Cytotoxicity Assays.
Cell-mediated cytotoxicity was determined by using a standard microcytotoxicity assay (21). In brief, target cells were pelleted and resuspended in 100 µCi of Na51Cr (DuPont-NEN, Boston, MA) per 106 cells and incubated at 37°C in a humidified 5% CO2 incubator for 1 h. They were washed three times with media, resuspended to 5 × 104/ml, and 0.1 ml was added to round-bottomed microtiter wells (Falcon Labware, Becton Dickinson & Co., Oxnard, CA). Varying numbers of effector cells were added in 0.1-ml vol to achieve the desired E/T ratios. For a spontaneous 51Cr-release control, 0.1 ml of media was substituted for effector cells. Maximum release was determined by adding 0.1 ml of 1% NP-40 (United States Biochemical Corp.) to the target cells. After 6 h at 37°C, the plates were centrifuged at 200 g for 5 min, and 0.1 ml of supernatant was removed from each well and counted on a gamma counter (model 5000; Beckman Instruments Inc., Palo Alto, CA). Data were presented as: percent specific 51Cr-release = 100 × [(experimental cpmOligonucleotides and Labeling.
The VRNA Extraction.
Total RNA was extracted from spleen cells, peritoneal exudate cells (PEC), and peripheral blood (PB) by the acid-guanidinium thiocyanate-phenol-chloroform method (23). In brief, <107 spleen cells or PEC were pelleted and subsequently extracted with 0.5 ml of solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, and 1% 2-mercaptoethanol) (Sigma Chemical Co.), 50 µl of 2 M sodium acetate, pH 4 (Sigma Chemical Co.), 0.5 ml phenol, pH 8 (United States Biochemical Corp.), and 0.1 ml chloroform (EM Science, Gibbstown, NJ)/isoamyl alcohol (Fisher Scientific Co.) (49:1). After a 15-min incubation on ice, the reaction mixture was centrifuged at 10,000 g for 20 min. The aqueous phase was transferred and subjected to two cycles of precipitation with 2-propanol (Fisher Scientific Co.). For PB samples, 0.25 ml PB (without anticoagulant) was mixed with 0.5 ml solution D and 75 µl 2 M sodium acetate and subjected to RNA extraction as described above. When RNA was extracted from a small amount of lymphocytes such as PB RNA samples were washed twice with 70% ethanol after propanol precipitation to remove excess salt, which otherwise would inhibit the RT-PCR reaction.CDR3 Length Spectratyping.
RNA samples, equivalent to 5-10 × 105 cells or 0.12 ml of blood, were amplified by using a GeneAmp RNA PCR kit (Perkin-Elmer Corp., Branchburg, NJ) with VDirect Sequencing of PCR Products.
RT-PCR products in 30- 75 µl were purified by using a QIAquick PCR purification kit (QIAGEN Inc., Santa Clarita, CA) and subjected to DNA sequencing by using specific unlabeled JStatistical Analysis.
To determine the significance of the preservation of a dominant peak in the memory response, we compared the proportion of the peak within that specific J ![]() |
Results |
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To study the evolution
of TCR repertoire usage in LCMV infection, we chose to
analyze the V8 TCR, because it is expressed on a significant number of T cells in the C57BL/6 mouse (4), and because the LCMV-specific lysis is highly represented within the V
8 population (19). As shown in Fig. 1, acute LCMV
infection expanded the CD8+V
8+ cells from 3 to 16% of
spleen cells or from 20 to 30-35% of total CD8+ cells.
Therefore, the CD8+V
8+ cells contributed greatly to the
major expansion of the CD8+ population during acute
LCMV infection. To characterize the specificity of these
CD8+V
8+ cells, we tested their cytolytic activities against
targets infected with LCMV or with the heterologous virus, PV. As shown in Table 1, the CD8+V
8+ cells induced during the acute LCMV infection specifically lysed LCMV-infected but not PV-infected targets. The CD8+
V
8+ cells also specifically lysed RMA-S cells pulsed with
LCMV peptides GP33, GP276, and NP396. We have reported previously that RMA-S cells pulsed with peptides
from other viruses are lysed much less by LCMV-induced
T cells (20). Interestingly, enrichment of specific CTL activities against all three LCMV immunodominant epitopes was detected within the CD8+V
8+ cells, indicating that
this subset of T cells is useful for examining the dynamics of
a complex T cell response directed against several epitopes
(Table 1).
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We next examined the contribution of the V8.1 and 8.2 subsets to the expansion of the V
8 population during acute
LCMV infection by using RT-PCR with the V
8.1- and
8.2-specific primers in combination with the C
primer. A
much stronger band was detected when the acute LCMV-infected splenic RNA was amplified with the V
8.1 and C
primers (data not shown). Thus, analysis of the V
8.1
TCR usage would shed light on the evolution of a significant part of the LCMV-induced T cell repertoire.
To determine the LCMV-
induced V8.1 TCR repertoire usage directly ex vivo, we
analyzed the TCR usage by CDR3 length spectratyping. Pilot experiments were performed by using five J
primers
to determine the spectratype during acute LCMV infection. Whereas naive mice generated the typical Gaussian
distribution (see Fig. 5, day 0), spectratypes were skewed in
LCMV-infected mice (Figs. 2 and 3). The LCMV-induced
splenic spectratypes were different among individual mice,
although there were some similarities. To confirm that the variation between spectratypes was due to the differences
of TCR usage among individual mice instead of problems
with the reproducibility of the technique, we took two different RNA samples and performed spectratyping in triplicates, beginning with separate RT-PCR reactions. The
spectratypes were highly reproducible within each RNA
sample (data not shown).
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Because the spectratypes varied between individual mice, studies on the evolution of the repertoire necessitated following the same individual mice longitudinally during LCMV infection, as a comparison of spectratypes between the mice would be impossible to interpret. This could only be done by examining PB spectratypes, as examining spleen cell spectratypes would require killing the mice. To see whether the PB spectratypes were representative of those in other immunological compartments, we compared the spectratypes in spleen, PB, and PEC after LCMV infection and found them to be nearly identical (Fig. 2). Fig. 2 shows the spectratypes for day 8 after infection, and spectratypes similar for spleen, PB, and PEC were also observed at days 6 and 12 after infection (data not shown). Thus, it was feasible to study the evolution of TCR repertoire usage in LCMV infection by determining the PB spectratype.
Acute LCMV Infection Expands a Broad Spectrum of VTo examine the complete V8.1+ T
cell repertoire in acute LCMV infection, we analyzed the
V
8.1 spectratypes with primers specific to all 12 J
s. The
V
8.1 spectratypes from naive mice at 6 wk and 4 mo of
age invariably had a normal Gaussian distribution for each
of the J
s (data not shown). In contrast, the acute LCMV infection expanded a broad spectrum of V
8.1+ T cells.
Dominant peaks were consistently detected in the spectratypes of J
s 1.1, 1.2, 1.3, 1.5, and 1.6 in most of the
mice tested; some other peaks occasionally were detected
in the J
2s (Fig. 3; The two types of graphic displays reflect
the fact that some analyses used 33P-labeling [Fig. 3 A] and
others, done later in the study, used fluorescent labeling
[Fig. 3 B]). The spectratypes determined by J
s 2.2 and 2.4 were consistently very weak, as the radioactivity intensities
(Y-axes) were low in both acutely infected and naive mice.
Thus, genetically identical hosts generate somewhat predictable but nevertheless different T cell spectratypes in response to the same virus. Of the 12 J
s, J
1s were preferentially used. This preferential expansion was elicited by
the virus infection and not by the diluted culture media vehicle, as the spectratype from vehicle-injected mice had a
normal Gaussian distribution (data not shown). The V
8.1
spectratype in acute PV infection, which also activated
V
8+ T cells, although to a lesser extent, was unlike that
observed in the acute LCMV infection, as PV infection did
not generate consistent dominant peaks in the spectratypes
of V
8.1-J
1.1 and V
8.1-J
1.2 (data not shown). The
observation of discrete LCMV-induced clonal expansion
by spectratyping indicates that the massive proliferation of
T cells within the V
8.1 population is unlikely due to
LCMV-encoded superantigen stimulation or nonspecific
cytokine stimulation.
To evaluate the contribution of the
CD4+ and CD8+ T cell populations to the skewed V8.1
spectratype during acute LCMV infection, we depleted
CD4+ T cells from the spleen cells at 9 d after LCMV infection and compared the spectratype of the CD4-depleted
spleen cells to that of the total spleen cells. As shown in Fig.
4, experiment 1 (EXPT. 1), the spectratype of the CD4-depleted spleen cells was very similar to that of the total
spleen cells, indicating that CD4+ T cells contribute little
to the skewed V
8.1 spectratype. We also sorted CD4+
and CD8+ T cells from the spleen cells at 8 d after LCMV
infection and determined their V
8.1 spectratype. The
CD8 spectratype was nearly identical to that of the unsorted spectratype (Fig. 4, EXPT. 2). Even though responses could be seen within the J
1.2 and J
1.3 CD4
spectra, in the total T cell population they were not sufficient to influence the more dominant response associated
with the CD8+ cells. Taken together, the skewed V
8.1
spectratype is dominated by the CD8+ T cell population,
and studies of the LCMV-induced V
8.1 spectratype by
using PB or spleen reflect mainly the LCMV-induced CD8
spectratype.
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The fact that LCMV-induced spectratypes differed between individual mice indicated that studies on the evolution of the T cell response could not be done by comparison of one mouse to another, but instead required longitudinal studies within the same mouse. We were able to do this by examining PB spectratypes. From days 10 to 14 after infection, a time period immediately after the peak in the massive CD8+ T cell proliferation, the majority of the T cells undergo apoptosis in vivo (11). Most virus is cleared by ~7 d after infection, and we asked whether there is a further selection of T cell receptors after viral clearance and during this apoptosis phase. Fig. 5 shows the PB spectratype from individual mice at days 6 to 12 after infection and compares it with the normal Gaussian distribution pattern of separately examined uninfected mice. The naive spectratype began to change by day 5 (data not shown) and evolved into a distinct spectratype by day 8 after infection. This distinct spectratype remained stable from days 8 to 12 after infection, even though there was a significant decrease in the number of T cells after day 9. This suggests that after viral antigens are cleared, further selection of the T cell repertoire does not occur during the T cell apoptosis period.
Sequence Analyses Show Stability of the TCR Repertoire within an Individual Mouse but Distinctions among Mice. To
determine whether a discrete spectratype peak was predominantly monoclonal and whether a further selection of
the TCR sequences occurred from days 8 to 12 after
LCMV infection, we directly sequenced PCR DNA amplified from day 8 and day 12 LCMV-infected PBL (Fig. 5)
by using J primers that had revealed singular and dominant spectratype bands. Fig. 6 shows that this technique
revealed predominantly monoclonal TCR sequences in
some mice. The sequences of mouse 4 were monoclonal,
whereas the sequences in mice 1 and 3 each appeared to
reveal two clones. The sequences in the CDR3 region remained unchanged from days 8 to 12 after LCMV infection in mice 1, 3, and 4. In mouse 2, it was hard to draw
any conclusion by comparing the sequences between day 8 and day 12 of LCMV infection because too many unreadable sequences were present in the CDR3 region, indicating that several clones were present. Surprisingly, the DNA
sequences were very different among these four mice even though they had a very similar J
1.6 spectratype with discrete, similarly sized bands. Notably, a conserved GXXN
amino acid motif with a non-V, non-J-encoded glycine
residue at position 96 was detected in all the sequences analyzed, even those from the multiclonal mouse 2, suggesting the importance of this motif for the recognition of
LCMV. These DNA sequence data further support the
concepts that genetically identical hosts generate different
T cell responses to the same antigen and that after 7 d of
LCMV infection, when virus is cleared, no further selection of the T cell repertoire occurs.
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A high dose LCMV clone 13 infection results in a transient antiviral CTL response followed by clonal exhaustion of the virus-specific T cells,
due to the overwhelming amount of antigen present (7). We asked how global the T cell exhaustion was by examining the V8.1 spectratype. As shown in Fig. 7 A, the
spectratype changed continuously from 7 to 13 d after
LCMV clone 13 infection, when excess viral antigens were
present. Some dominant peaks in the V
8.1 spectratype
disappeared at days 10 or 13 after infection: mouse 1 J
1.1,
mouse 3 J
1.2, mouse 4 J
1.3, mice 1 and 4 J
1.5, and
mouse 3 J
1.6. Others changed from 7 to 13 d after infection, accompanied with new peak formation: mouse 2 J
1.1, and mice 2 and 4 J
1.6. However, others remained
stable, such as those of mice 1 and 4 J
1.2, mouse 3 J
1.3,
and mouse 1 J
1.6. To determine whether all of the virus-induced CD8+ T cells were clonally exhausted and whether
the skewed spectratype was due exclusively to CD4+ T
cells, we sorted CD4+ and CD8+ cells from the spleen cells
at 13 d after LCMV clone 13 infection to determine their
V
8.1 spectratype. As shown in Fig. 8, skewed spectratypes
were detected in both CD4+ and CD8+ T cell populations. Therefore, the change of spectratype from 7-13 d after LCMV clone 13 infection was not due to selective preservation of the CD4+ T cell population.
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To investigate whether a subset of T cells activated by
high dose clone 13 infection was resistant to clonal exhaustion and survived in persistent infection, we examined the
V8.1 spectratype during longer term infections. Although
we still detected skewed V
8.1 spectratypes at day 15 and,
to a lesser extent, at day 22 after infection (data not shown),
almost all of the spectratypes became like those of naive
mice by 42 d after infection and remained the same thereafter (Fig. 7 B). Only very weak non-Gaussian spectratypes could sometimes be detected, such as that of J
1.6 in
mouse 5 (Fig. 7 B). This observation suggests that most
clone 13-induced T cells eventually underwent clonal exhaustion. In summary, these results support the concept
that continuous selection of T cells occurs in the presence
of persistent antigens, but they also show that T cell clonal
exhaustion occurs in the great majority of the virus-
induced T cells, and only very weak T cell responses can be
detected long after infection.
It has not
been known which activated T cells and what proportions of
these T cells survive the apoptosis phase and become memory cells. Our LDA data indicate that there is only a two-fold reduction in the numbers of LCMV-specific pCTL
per CD8+ cell between the peak of the acute infection and
long term memory. The efficiencies of these assays, which
indicate that ~2% of the CD8+ T cells are precursors for
LCMV-specific CTL in immune mice, are unknown, and
a much higher proportion of the T cells in the immune
state may be specific for the virus than previously thought. A recent study on tetrameric MHC-peptide staining of
TCR supports the concept that a substantial number of
memory cells remain virus specific (13). To determine the
preservation and distribution of virus-induced T cell species in the memory state, we compared the V8.1 spectratype in the acute LCMV infection with that in the immune state from the same individual mice. The immune
spectratype (Fig. 9 A, day 42; Fig. 9 C, 2-2.5 mo) was very
different from the naive spectratype but resembled a combination of that of acutely infected and naive mice, consistent with only a moderate dilution of memory cells with
naive cells (Fig. 9 A). This supports the concept that
LCMV infection alters the host T cell repertoire dramatically and leaves it with a high frequency of LCMV-specific T cells (9). We found that very dominant peaks in the
acute infection often remained present in the immune
state. Six dominant peaks consisting of ~70% of the T cells
in their specific J
spectratypes were selected for sequence
analysis. By direct sequencing of the PCR DNA, we were
able to resolve a monoclonal sequence from one of the six
peaks. Its TCR sequence was identical to that seen at day
10 of the acute infection (Fig. 6), and, notably, it had the
same GXXN conserved motif seen in the acute J
1.6 samples. It appears that very strong dominant peaks in the acute
infection, regardless of which J
they use, can be detected
in the immune state by spectratyping.
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Although pCTL data have shown that a high frequency
of LCMV-specific pCTL remains stable throughout the
lifetime of the mouse (1), it was still not clear how the
TCR repertoire evolved long after infection and whether
such a high frequency of pCTL reflected a drastic alteration
of the host T cell repertoire in long term memory. Therefore, we examined the evolution of V8.1 TCR repertoire
into long term memory by spectratyping. As shown in Fig.
9 B, dominant peaks were stably preserved from acute
LCMV infection through 7 mo after infection, and during
this period of time no other new peaks were generated.
Thus, a high frequency of LCMV-induced T cells can be
preserved from acute infection into long term memory and
permanently skew the host T cell repertoire.
In the immune state, the spectratypes of PB and spleen were similar to each other, but that of PEC was somewhat different (Fig. 9 C) in that it even more closely resembled that of the PB during the acute infection, which is consistent with our previous findings that LCMV-immune PEC contain a higher frequency of LCMV-specific pCTL (25). This might be because the LCMV infection had been established previously by i.p. inoculation or because few naive T cells accumulate in the peritoneal cavity to dilute out the memory cell population.
To determine the effects of secondary LCMV infection
on the spectratype, we rechallenged the immune mice with
a high dose of LCMV (107 PFU) and then analyzed their
V8.1 spectratypes. The LCMV-induced secondary spectratype was similar in most of the dominant peaks but was
different in some, compared with that of the primary infection. Fig. 9 A shows that subdominant peaks, such as those
of J
1.5 in mice 1 and 2 and that of J
1.6 in mouse 1, appeared in the secondary response for the first time. The further
selection of the TCR repertoire was not due to a nonspecific activation induced by culture media, as the media-injected
LCMV-immune mice had a spectratype similar to that of
their immune state (data not shown). To confirm that the
secondary infection activated LCMV-specific T cells, we
examined CTL activities on the same day (day 5) as the
spectratype analyses after secondary LCMV infection. Significant LCMV-specific CTL activities were detected (63%
specific lysis against LCMV-infected targets at E/T = 200 in a direct ex vivo 6-h assay). These results support the
generally accepted concept that further maturation of the T
cell response can occur in a secondary viral infection.
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Discussion |
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In this study we have provided evidence supporting the
concepts that (a) the virus-induced T cell repertoire during
a primary infection changes little after clearance of the viral
antigens; (b) it is continuously selected in the presence of
excess viral antigens leading to persistent infection; (c) the
LCMV infection substantially skews the host T cell repertoire in the memory state long after virus is cleared; and (d)
genetically identical mice generate somewhat predictable
but nevertheless remarkably different T cell responses to
the same antigens. Evidence supporting the first and second
concepts is that the LCMV-induced V8.1 spectratype changes little between 8 and 12 d after LCMV infection, a
time when the virus has already been cleared (Fig. 5), but it
continues to evolve from 7 to 13 d after the infection of a
high dose of the clonally exhausting LCMV clone 13 (Fig.
7 A). Our spectratype data are consistent with our kinetic
studies on the pCTL frequencies, which show that the proportions of the three LCMV immunodominant peptide-
specific pCTL become fixed within the LCMV-specific
repertoire by day 7 after infection, and thereafter remain
the same throughout the lifetime of the mouse (9). Although a limitation of the spectratyping technique is that it
cannot detect subtle changes of the TCR usage within each
peak of the spectratype, our direct sequencing of the PCR
DNA revealed that the TCR sequences of the same-sized
dominant peaks within a specific J
spectrum induced by
the parent LCMV strain remained stable between days 8 and 12 after infection (Fig. 6). In contrast, the spectratype
analyses showed that from 7 to 13 d after LCMV clone 13 infection the spectratypes changed continuously (Fig. 7 A);
such changes were not due to selective preservation of
CD4+ T cells, as skewed spectratypes were detected in
both CD4+ and CD8+ T cell populations at 13 d after infection (Fig. 8). The disappearance of the dominant peaks
from 7 to 10 d of the clone 13 infection paralleled the deletion of the virus-specific CTL precursors; such a reduction
in pCTL was originally reported by Moskophidis et al. (7)
and confirmed by us during the course of these studies (26).
The appearance of new peaks and the stable presence of other peaks from 7 to 13 d after clone 13 infection indicated that some T cells remained activated instead of undergoing clonal exhaustion during this period of time.
However, the majority of these T cells eventually underwent clonal exhaustion long after the initial infection by
continuous stimulation with excess viral antigens, as most
spectratypes became like those of naive mice; only very
weakly skewed spectratypes could occasionally be detected at 42 d after clone 13 infection (Fig. 7 B). Taken together,
these results suggest that no further selection of LCMV-specific T cells occurs during the apoptosis phase in acute
LCMV infection, although a continuous selection of LCMV-specific T cells does occur in the presence of excess viral
antigens in LCMV clone 13 infection.
A previous, unresolved question was to what degree the specific immune response contracts after a systemic virus infection. Our spectratype data show that the LCMV infection substantially skews the host T cell repertoire in the memory state, as dominant peaks from the acute infection remain present. The immune spectratype 6-8 wk after infection is very different from that of naive mice, but resembles the combination of that of acutely infected and naive mice (Fig. 9 A). Thus, a host's T cell repertoire can be substantially altered by a virus infection, and remains skewed after the virus has been cleared. This molecular analysis is consistent with previous observations revealed by LDA and by quantification of transgenic T cells showing that LCMV infection leaves the host T cell repertoire with a very high frequency of LCMV-specific T cells (9, 12, 27), a claim also supported by a recent report on MHC-tetramer binding studies. However, here we have been able to show how a multiclonal T cell repertoire evolves from acute infection and is preserved in the memory state.
pCTL data have shown that the frequency of the
LCMV-specific pCTL remains stable throughout the lifetime of the mouse (9, 12). In this study, we have examined
the preservation of the LCMV-induced V8.1 spectratype
up to 7 mo after infection. We found that the relative intensity of a dominant peak within that specific spectrum
was weaker at 4 mo than at 6-7 wk after LCMV infection
(data not shown), but thereafter remained stable at least
through 7 mo of infection (Fig. 9 B). This may reflect the ability of RT-PCR to measure mRNA levels, which were
probably reduced in the older memory cells, as incubation
of the immune spleen cells or PB leukocytes with IL-2 rapidly revealed a spectratype similar to that of the acute infection (data not shown).
A broad spectrum of V8.1+ T cells was activated during acute LCMV infection (Fig. 3). A previous report examining specific T cell clones to Epstein-Barr virus suggested that an immunodominant epitope induced a broad
T cell repertoire and that a subdominant epitope induced a
more restricted T cell repertoire (28). The V
8+ T cells
recognize three LCMV immunodominant peptides (Table
1), so it is not surprising that a broad spectrum of T cells
were activated during acute LCMV infection. Since dominant peaks in the acute infection are present in the immune
state, it also appears that a broad repertoire of LCMV-specific T cells can be selected into memory. Some CTL
clones isolated from LCMV-immune mice infected with
serologically unrelated viruses, such as vaccinia virus or PV,
cross-react between the two viruses (29). A broad spectrum of LCMV-specific memory T cells enhances the probability of having cross-reactive memory cell clones that can be
stimulated and expanded quickly in the event of a second
virus infection, thus providing protective immunity or influencing the pathogenesis of a second infection. Experiments have, in fact, demonstrated that a history of LCMV
infection provides protective immunity against vaccinia virus,
PV, and murine cytomegalovirus by controlling the viral replication early in infection (1). This examination of the V
8.1
TCR usage suggests that the majority of the dominant T cell clones detectable by spectratyping is not eliminated during the apoptosis phase and survive in the memory cell population.
The fact that the spectratypes of immune splenocytes and PB resemble a combination of the spectratypes found in these compartments of acutely infected and naive mice suggests that virus-specific memory cells become diluted with naive cells, a result consistent with our LDA analyses (9). However, the immune PEC have a spectratype virtually identical to that of the lymphoid compartments during the acute infection, which is consistent with the observation that immune PEC contained a very high frequency of LCMV-specific T cells (25). This result could be due to the fact that there is a lack of dilution of the memory cells with naive cells in the peritoneal cavity. Alternatively, the LCMV-specific memory T cells may be more likely to circulate back to the initial peritoneal cavity inoculation site. It is also possible that some viral antigens remain in the peritoneal cavity to provide continuous stimulation to the LCMV-specific T cells, such that they express a higher level of TCR RNA.
A recent study of the functional fine specificity of the LCMV-specific CTL response led Bachmann et al. (30) to propose that many T cells were deleted from the repertoire during the apoptosis phase of the primary LCMV infection. Previously published LDA data (9) as well as our data here showing that the spectratype and the examined TCR sequences remained stable during the apoptosis phase of the primary infection (Figs. 5 and 6) are inconsistent with that suggestion. However, they do not rule out the possibility that weak T cell responses insufficient to register in LDA or spectratype analyses were eliminated during this time period. Nevertheless, our data would indicate that changes in the T cell repertoire between the primary and secondary infections are not a consequence of the apoptotic phase of infection, but instead are due to the selective pressure of antigen during secondary infection.
An observation that we found of great interest is that
even though genetically identical mice generate similar levels of CTL responses against the LCMV immunodominant
peptides (9, 13), the TCR repertoire generating these responses differed from mouse to mouse (Fig. 3). Differences
in spectratypes were even seen in LCMV-infected, /
TCR-deficient mice reconstituted with a common pool of
euthymic mouse splenocytes, supporting the argument that
these differences were not caused by prior exposure to different antigens by the individual mice (data not shown).
C57BL/6 mice infected with the same dose of LCMV had
different spectratypes (Fig. 3). There was preferential J
usage and some homogeneity in the CDR3 length, but sequence analysis of the same sized discrete V
8.1-J
1.6
spectratype peaks revealed different DNA sequences. Although the DNA sequences were different, a conserved
GXXN amino acid motif was detected in all the sequences
analyzed, suggesting the importance of both the CDR3
length and this motif for the recognition of LCMV. Direct
sequencing of dominant spectratype bands may be a useful
technique to define conserved motifs associated with regions of the TCR involved in binding to the peptide-
MHC complex. These observations are complementary to
that of Maryanski et al. (31), who found that each of the
mice immunized with the P815-CW3 cells displayed distinct but structurally similar CD8+ TCR repertoires to the
HLA-CW3 170-179 epitope, in that they had similar
CDR3 length and displayed a conserved non-V, non-J- encoded glycine residue. A stochastic effect may play an
important role in the differences of T cell responses to the
same antigens. The fact that genetically identical hosts generate different T cell responses to the same antigens may
help explain the variation of incidence in virus-induced autoimmune diseases in genetically identical hosts. For example, ~30% of the diabetes-resistant BB/Wor rats developed
diabetes after Kilham's rat virus infection (32).
In summary, the data presented here indicate that genetically identical hosts generate remarkably different T cell responses to the same antigens and that studies on the evolution of the T cell repertoire require following the same individual hosts longitudinally during an antigenic challenge. Data from such a longitudinal study support the concepts that the virus-induced T cell repertoire remains stable both after clearance of the virus and during the apoptosis phase of the T cell response but evolves in the presence of the viral antigens, as shown in the clone 13 model and during secondary LCMV infection. A multiclonal T cell response induced during an infection is preserved into memory and continues to markedly skew the host T cell repertoire long after the virus has been cleared.
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Footnotes |
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Address correspondence to Dr. Raymond Welsh, Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Phone: 508-856-5819; Fax: 508-856-5780; E-mail: RWelsh{at}bangate.ummed.edu
Received for publication 6 April 1998 and in revised form 18 August 1998.
1 Abbreviations used in this paper: CDR3, complementarity-determining region 3; EXPT, experiment; GP, glycoprotein; LDA, limiting dilution assay; LCMV, lymphocytic choriomeningitis virus; NP, nucleoprotein; PB, peripheral blood; pCTL, precursor cytotoxic T lymphocyte; PEC, peritoneal exudate cell; pTh, precursor T helper cell; PV, Pichinde virus.We would like to thank Drs. Liisa K. Selin and Eva Szomolanyi-Tsuda for valuable discussions, Jie Yin for excellent technical assistance, and Phyllis Spatrick and Barbara Eddy for DNA sequencing.
This work was supported by National Institutes of Health grants AI-17672 and AR-35506 to R.M. Welsh.
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