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Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice were obtained from both The Jackson Laboratory (Bar Harbor, ME) and the Frederick Cancer and Research Development Center (Frederick, MD), while SCID mice (BALB/c background) were obtained from the Frederick Cancer and Research Development Center. P815 is a DBA/2-derived mastocytoma. 15-12RM is a transfected, tumorigenic BALB/c 3T3 cell line that expresses the gp160 protein from HIV-1IIIB as well as the ras and myc oncogenes (26, 27). 18neo is a similar control cell line transfected only with the neo vector. BSC-1 is an African green monkey kidney epithelial cell line that allows easy recognition of vaccinia plaques.
Peptide
Peptide p18-I10 (I10), the 10-aa (RGPGRAFVTI), immunodominant peptide epitope within HIV-1IIIB gp160 (26, 28), was obtained from Anaspec (San Jose, CA).
IFN-
ELISA
IFN- was measured in culture supernatants 24 or 48
h after CTL activation using a mouse IFN- minikit obtained from
Endogen (Cambridge, MA).
Recombinant vaccinia virus
vPE16 is a recombinant vaccinia virus that expresses the gp160 protein from the IIIB strain of HIV-1 (29) and was a kind gift from Patricia Earl and Bernard Moss (National Institute of Allergy and Infectious Diseases, Bethesda, MD). vSC8 is a similar control vaccinia construct that does not express gp160 (30). Stock solutions of virus were stored at -70°C; thawed virus was sonicated three times for 20 s at 4°C before making appropriate dilutions. Virus solutions were titered as described below. For infection of target cells in vitro, 1 ml of cell culture medium containing 5 x 107 PFU of vaccinia was added to 1 x 106 P815 cells and then incubated at 37°C until assayed.
In vivo CTL reconstitution and vaccinia titers
H-2d SCID mice were inoculated i.v. via the tail vein with varying amounts of CTL in 100 µl to 1 ml of PBS. CTL used in reconstitution experiments had been restimulated 4 or 5 days before. Immediately following CTL administration, 1–5 x 107 PFU of recombinant vaccinia were injected i.p. in 200 µl of PBS. The quantity of virus is critical since excessive viral titers obscure the protection of CTL adoptively transferred into SCID mice. Three or 6 days later, mice were sacrificed and tissues were harvested. Samples were frozen at -70°C until analysis for vaccinia PFU as per Buller and Wallace (31). Briefly, ovaries were frozen and thawed three times, homogenized in 1 ml of PBS, sonicated for 1 min, and spun briefly to obtain a clear supernatant containing virus. Serial dilutions of the supernatant were made in medium containing 2.5% FBS and 1 ml of each supernatant was added per well to monolayers of BSC-1 cells in a six-well plate. After 2 h, 2 ml of additional medium was added. Forty-eight hours later, the cell monolayers were fixed and stained, and the vaccinia plaques were evaluated to ascertain the PFU per ovary.
CTL homing and sectioning of ovarian tissue
CTL were labeled by incubation with 10 µM 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC16)3 (Molecular Probes, Eugene, OR) for 15 min at 37°C or 2.5 µM Cell Tracker Green (Molecular Probes) for 45 min at 37°C before washing in PBS before use. CTL (1 x 107) were injected into the tail vein of SCID mice, followed immediately by i.p. administration of 5 x 107 PFU of vPE16. Twenty-four to 48 h later, the ovaries were removed. If labeled with diIC16, they were embedded in OCT, frozen on dry ice, and stored at -70°C before sectioning with a cryostat. Ovaries labeled with Cell Tracker Green were fixed with 4% formaldehyde in PBS and processed for conventional paraffin embedding and sectioning. In either case, sections were analyzed by fluorescent microscopy and CTL were quantified by obtaining the mean ± SD of the total CTL in x400 high-powered fields of ovary sections from each of three separate experiments. These two methods are complementary: diIC16 requires visualization within frozen sections with corresponding suboptimal histology, whereas Cell Tracker Green can be visualized within paraffin sections, but against a high autofluorescence inherent to ovarian tissues.
Generation of CTL lines and clones
CTL lines were generated using either APC pulsed for 2 h
with peptide or the addition of free peptide to cocultures of CTL and
APC. Initial cultures were started using cells obtained from the
spleens of BALB/c mice immunized 1–3 mo previously with vPE16. For
lines generated with pulsed APC, 7.5 x 106
responding spleen cells were cocultured with 3.5 x
106 irradiated (3000 rad) stimulating BALB/c
splenocytes pulsed with various concentrations (1 x
102, 1 x 10-1, or
1 x 10-4 µM) of I10 peptide in a 24-well
plate containing 2 ml of RPMI 1640 medium supplemented with HEPES,
L-glutamine, sodium pyruvate, nonessential amino acids,
penicillin, streptomycin, 5 x 10-5 M 2-ME,
10% FCS, and 10% T-Stim (Collaborative Biomedical Products, Bedford,
MA). Lines generated with free I10 peptide were prepared similarly, but
had 5 x 10-5–5 x
100 µM free I10 added directly to the cultures.
CTL lines were established from primary cultures and were maintained in
24-well plates by weekly restimulation of 3–5 x
105 cells/well in the presence of 5 x
106 irradiated (3000 rad) BALB/c spleen cells
either pulsed with the appropriate concentration of I10 peptide or with
I10 peptide added directly to the culture. For experiments requiring
greater numbers of CTL, 1 x 107 CTL were
grown in T75 flasks in the presence of 0.5–1 x
108 irradiated spleen cells in 12.5 ml of medium
containing free peptide. CTL clones were established from CTL lines of
various avidities by limiting dilution cloning. Individual clones were
obtained from plates containing growth in <8 of 96 wells, screened for
uniformity of TCRV
usage, and maintained under conditions
appropriate for their avidity.
Avidity
CTL avidity was defined as the negative log of the peptide concentration that resulted in 50% maximal target cell lysis. CTL avidity was also assessed by determining susceptibility of target cell lysis to blockade by 10 µg/ml anti-CD8 (clone 53-6.7; PharMingen, San Diego, CA).
51Cr release and europium/bis(acetoxymethyl)2,2':6',2'-terpyridine-6,6'-dicarboxylate (BATDA) assays
The 51Cr release assay was conducted as
previously described (32). Europium/BATDA assays were
performed similarly. Target cells (1 x 106)
were either unpulsed or pulsed with an appropriate concentration of
peptide in culture medium containing 2 mM probenecid for 2 h at
37°C. A volume of 5 µl of BATDA was added during the last 15 min of
labeling. BATDA-loaded APC were washed five times in 12.5 ml of warm
culture medium containing 2 mM probenecid, with a 20 min rest between
the fourth and fifth washes. CTL were added at a 12.5:1 E:T ratio to
the peptide-pulsed, BATDA-loaded, and washed APC and incubated in
culture medium that also contained 2 mM probenecid. At one or more time
points, from 1 to 4.5 h, 20-µl samples of supernatant were added
to 200 µl of europium solution in a 96-well plate. Spontaneous
release of BATDA from P815 target cells prevented extension of assays
beyond 4.5 h. After shaking for 15 min, free BATDA released into
the supernatant by lysis of the target cells could be measured by the
time-delayed fluorescence of BATDA-chelated europium in a Wallac
Victor2 (Perkin-Elmer, Gaithersburg, MD). When
wells were sampled at only one time point, the specific lysis was
calculated identically to that for the 51Cr
release assay. Since europium assays allowed the same well to be
sequentially sampled to examine the kinetics of lysis, a modification
of the formula was used to account for the serial 20-µl reductions in
the 200-µl initial well volume after sampling at each time point. The
following formula was used to calculate the specific fluorescence of
sample n:
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTL lines were generated using either peptide-pulsed APC or by
addition of free peptide to the culture medium (22).
Regardless of the means used to generate them, CTL lines of a given
avidity exhibited similar properties. High-avidity cells could be
generated using either low concentrations of peptide pulsed onto APC or
by addition of low concentrations of peptide to the culture medium.
Very low-avidity cells, on the other hand, were best generated by
addition of high concentrations of free peptide to the culture medium;
equivalent high Ag densities could not be easily obtained using lines
generated with pulsed cells. Since previous work was all done with
uncloned CTL lines, for this study CTL clones were generated by
limiting dilution from high-avidity, low-avidity, and very low-avidity
CTL lines and found to always retain the properties of their parental
cell lines (Fig. 1
A). They
exhibited a 3–4 log10 difference in functional
avidity measured as peptide concentration necessary to produce either
50% lysis (Table I) or IFN- (Fig. 1B). Avidity was also confirmed by the sensitivity of
Ag-density requirements to anti-CD8 Ab (Ref. 22 ; data
not shown). Since all of the CTL lines and clones make IFN-, they
are all of a similar Tc1 phenotype.
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Previously, we demonstrated that high-avidity CTL, but not
low-avidity CTL, were effective at reducing viral titers in vivo
(22). This preferential reduction occurred despite the
ability of both high- and low-avidity CTL to recognize virally infected
cells in vitro. We hypothesized that the lower viral titers seen in
vivo after adoptive transfer of high-avidity CTL might be due to the
ability of these CTL to recognize and lyse target cells earlier in the
course of a cellular viral infection. At such times, just as viral
components were initially synthesized, the Ag densities would be so low
that only the high-avidity CTL would be able to recognize infected
cells. Correspondingly, low-avidity CTL, which require a high
peptide/MHC determinant density, would be unable to recognize and lyse
target cells until later in viral development when higher amounts of
peptide-MHC complexes would be displayed at the cell surface and viral
progeny were already likely to have been assembled. The hypothesis was
examined by contrasting the times at which high-and low-avidity CTL
were first able to recognize and lyse target cells infected with vPE16,
a recombinant vaccinia virus containing HIV-1IIIB
gp160. Lytic assays were initiated by addition of CTL to P815 target
cells at various times after infection of the target cells with vPE16
virus, and the total target cell lysis was assessed after a 4-h
incubation. As shown in Fig. 2, high-avidity CTL recognized vaccinia-infected targets as early as 2–4
h after infection with vPE16. In contrast, low-avidity CTL could
recognize targets only at later time points. Furthermore, even though
both high- and low-avidity CTL were able to similarly recognize and
lyse peptide-pulsed target cells presenting very high Ag densities
(indicating a similar lytic activity if Ag density became sufficiently
high), high-avidity CTL were much more effective in killing
virus-infected cells up to 22 h after the onset of infection (Fig. 2). In particular, the slopes of the kinetic curves for target cell
lysis are substantially different during the first 8 h. These
results support the hypothesis that high-avidity CTL recognize
virus-infected cells earlier in the time course of infection than do
low-avidity CTL, and thus prevent an increase in viral titer by killing
infected cells before the assembly and release of new virus.
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). All CTL clones could recognize
I10-pulsed 18neo control cells, but only the high-avidity clone was
able to recognize and lyse both the 15-12RM cells and the I10-pulsed
18neo control cells equally well. The low-avidity clone recognized the
15-12RM cells with a lowered efficacy, whereas the very low-avidity 6Aa
clone did not lyse the 15-12RM cells at all, even though it was capable
of recognizing the 18neo cells presenting high peptide-Ag densities.
These results confirm that high-avidity CTL can recognize Ag presented
endogenously at densities not seen by lower avidity CTL, and further
support the hypothesis that high-avidity CTL are capable of recognizing
and killing cells earlier in the course of viral infection.
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The previous results indicated that high-avidity CTL could
recognize the low Ag densities resulting from endogenous vPE16
infection earlier than low-avidity CTL, but since these results were
obtained using 51Cr lytic assays that represented
the total lysis present after 
4 h of incubation, they did not address
differences in the rate of lysis. It remained possible that
high-avidity CTL not only recognized lower Ag densities, but also
killed target cells at a higher constant rate. The kinetics of lysis of
targets pulsed with a fixed amount of peptide by CTL of varying avidity
is distinct from the kinetics by which infected targets become
susceptible to lysis as increasing levels of viral protein are made, as
was described in Fig. 2
. To examine this question, we used the Europium
lytic assay to compare the ability of four clones of differing avidity
to lyse target cells presenting high Ag density. As seen in Fig. 4A, when targets were pulsed
with 10 µM I10 peptide, a concentration allowing lysis by all of the
CTL, high-avidity CTL began to lyse the target cells immediately, but
lower avidity CTL revealed a delay in the onset of their lytic
activity. Once lysis began, however, the lower avidity CTL killed
target cells at a rate similar to that of the higher avidity CTL. Due
to the relationship between CTL avidity and Ag density, we also
examined the lytic kinetics of high-avidity CTL over a range of Ag
densities. Fig. 4B shows that as the Ag density decreased to
a range below that detectable by the lower avidity CTL (1 x
10-3 µM), the high-avidity CTL showed an
increasing delay in their activation. As seen above with the
low-avidity CTL, however, once lysis was initiated, the rates of lysis
were similar. Thus, high-avidity CTL initiate target cell lysis more
quickly at Ag densities at which both high- and low-avidity CTL can
respond, but they also initiate target cell lysis, albeit not as
quickly, at densities at which low-avidity CTL do not function at all.
Therefore, a second mechanism by which CTL may more effectively reduce
viral burden is their ability to initiate lysis earlier at a given
level of target Ag expression and thus kill more infected target cells
in a shorter time.
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Since we had previously demonstrated that low-avidity CTL lines
are less effective in reducing viral burden in vivo than equivalent
numbers of high-avidity CTL (22), but had never
quantitatively compared their potency in vivo by titrating the numbers
of cells transferred, we asked whether transferring increased numbers
of low-avidity cells could compensate for their lower efficacy. As is
shown in Fig. 5A, increasing
the number of low-avidity CTL to 3 x 107,
the maximum number of low-avidity CTL reasonably generated for adoptive
transfer with our culture system, still produced little reduction in
viral titer. On the other hand, we were surprised to find that a 3-fold
reduction in the number of high-avidity CTL transferred (from 1 x
107–3.3 x 106)
significantly reduced their effectiveness (Fig. 5A). We
extended these observations by repeating this experiment using high-,
low-, and very low-avidity CTL clones. As seen in the earlier
experiment using CTL lines, the high-avidity CTL clone showed a sharp
decrease in effectiveness with a small drop in the number of cells
transferred (Fig. 5B). CTL from the low-avidity 1 Da clone,
which corresponded most closely in avidity to the low-avidity line used
in the first experiment, showed a small decrease in viral titer at the
highest concentration transferred. The lowest avidity clone, on the
other hand, showed no reduction in viral titer at any concentration
examined. Since effective clearance of vaccinia virus in vivo has been
ascribed to IFN- (33), we examined the levels of
IFN- produced by CTL clones and lines of various avidities. Although
the Ag densities required for cytokine production reflected the CTL
avidity (Fig. 1B and Refs. 34, 35), there
were no consistent associations of IFN- production with CTL avidity
because, in general, IFN- production at high Ag density was roughly
equivalent (Table II). In summary, either
high-avidity clones or CTL lines produced a response curve in vivo
shifted more than a log to the left of low-avidity clones or CTL lines,
indicating more than a 10-fold greater efficiency in reduction of viral
titers in vivo.
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In the previous experiments in which low-avidity CTL were shown to
be ineffective for viral clearance, only a single time point (3 days
after infection) was analyzed. Given that low-avidity CTL showed a
delayed and slower lytic rate in vitro and transferring larger numbers
of low-avidity CTL increased their ability to lower viral titer (see
Fig. 5), the possibility remained that low-avidity CTL were capable of
eliminating virus in vivo but did so with slower kinetics. To determine
whether this was indeed the case, we compared the viral burden 3 and 6
days after transfer of high- and low-avidity CTL. Fig. 6 shows that these low-avidity CTL remain
incapable of reducing viral titers even when given a longer time to
act. Thus, the lack of clearance is an absolute and not a kinetic
difference between high- and low-avidity CTL in vivo.
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The increased effectiveness of high-avidity CTL to reduce viral
titers after infection could potentially be due either to their ability
to home to the site of infection earlier than low-avidity CTL or to
their ability to lyse infected cells earlier during the course of
infection. To distinguish between these alternatives, we examined
whether high-avidity CTL home to the site of infection earlier than
low-avidity CTL. Following the same procedure employed for adoptive
transfer experiments, 1 x 107 CTL labeled
with either lipid-permeable diIC16 or cytoplasmic
Cell Tracker Green were injected into the tail vein of SCID mice which
simultaneously received 5 x 107 PFU vPE16
i.p. After 24 or 48 h, ovaries were harvested and sectioned to
compare the efficiency of homing of both high- and low-avidity CTL.
Examination of histological sections showed no significant difference
between the numbers of high- or low-avidity CTL that were irregularly
distributed throughout the ovary, primarily in the ovarian stroma, by
24 h after infection (Fig. 7).
Similar results were seen at 48 h (although increasing
inflammation and vesicle formation in control ovaries and those
receiving low-avidity CTL made histology more difficult), and when
ovary cells were dispersed and examined by flow cytometry (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we used peptide-pulsed APC to generate high- and
low-avidity CTL lines. High-avidity CTL resulted from expansion using
APC pulsed to present low Ag densities, whereas low-avidity CTL
resulted from CTL exposed to APC presenting high Ag densities. In an
attempt to simplify and expedite the production and maintenance of such
lines, we generated a series of lines against various concentrations of
free peptide. It was possible to generate equivalent CTL lines by using
either APC pulsed with peptide or by using free peptide added to
cocultures of CTL and APC, but although all high-avidity CTL shared
similar properties, as did low-avidity CTL, the concentrations of
peptide required to generate a given avidity varied between the two
approaches. We also isolated clones from CTL lines with a range of
avidities to avoid problems inherent with the use of a population of
cells that shared a common avidity, but which might differ in their TCR
affinity. Despite the potential variability of CTL lines, clones
generated from a specific line shared nearly identical avidity with the
parental line (Fig. 1A).
In our previous study, we demonstrated that a fixed number of
high-avidity CTL was much more effective than the same number of
low-avidity CTL in reducing viral titers in vivo (22), but
we did not compare the CTL quantitatively by titration in vivo. The
current study was designed to elucidate the mechanism underlying this
increased efficacy. Here, we show that lower avidity CTL remained less
capable of reducing viral burden, even when the number of transferred
CTL is increased 3-fold or when more time is allowed for clearance.
This was perhaps surprising since both high- and low-avidity lines
could recognize virally infected targets in vitro (22). We
hypothesized that the mechanism underlying the lack of effectiveness in
vivo was a result of the amount of viral protein required to obtain the
threshold determinant density necessary for recognition by low vs
high-avidity CTL and the efficiency by which high-avidity CTL could
then eliminate infected cells. Previous investigators have postulated
that CTL that can recognize target cells early in infection, when the
density of viral peptide-MHC complexes is still low, may be more
effective in clearing virus infection than CTL that can kill only
targets already loaded with high levels of viral progeny
(36, 37, 38). In contrast, low-avidity CTL requiring an Ag
density so high that it is achieved just before the release of
assembled virus from a cell are unlikely to be as effective in the
control of viral titers, even though as the infection progressed and
viral titers increased, their efficacy would undoubtedly improve. In
Fig. 2, we show that high-avidity CTL are more capable than low-avidity
CTL of recognizing target cells early in the course of viral infection,
as seen by the substantial difference in slopes within the first
8 h.
Further evidence that determinant density controls the recognition of
infected cells is provided by the CTL lysis of 15-12RM target cells.
15-12RM cells endogenously present low levels of I10 Ag, as the gp160
protein cannot be detected by Western blot even though the mRNA can be
detected (26). Only high-avidity CTL can recognize this
cell line as effectively as they recognize control 18neo cells pulsed
with peptide. Low-avidity CTL are less effective, and the lowest
avidity CTL do not recognize the 15-12RM cells at all, despite being
able to recognize and lyse 1) P815 target cells infected with vPE16
recombinant vaccinia virus and 2) control target cells pulsed with high
concentrations of peptide Ag as effectively as high-avidity CTL. In
vivo, it is not clear whether CTL actually clear virus directly by
lysis of infected cells or indirectly through the action of cytokines
such as IFN- (33). Regardless, since cytokine
production and lytic activity are equally correlated with avidity
(Refs. 34, 35 and Fig. 1B), either could
account for the observed differences in efficacy.
These observations support the hypothesis that the amount of time
required for infected cells to present an adequate determinant density
is a limiting factor for lysis of infected cells by low-avidity CTL and
that the delay incurred with the low-avidity CTL allows for the
production of increasing amounts of assembled virus and a perpetuation
of the infection. Furthermore, as we observed in Fig. 4, this
difference is compounded by the fact that, in contrast to high-avidity
CTL, low-avidity CTL exhibit a delay in the onset of their lytic
activity, presumably due to inherent differences in the efficiency of
TCR signaling. Once initiated, the rates of lysis seem equivalent, but
any delay in the onset of lysis only provides more time for viral
proliferation and contributes to the reduced effectiveness of
low-avidity CTL in combating viral infections. Further evidence
indicating that this delay is inherent in the TCR signaling efficiency
appears at very low Ag densities when high-avidity CTL also show a
delay in the onset of lytic activity. Taken together, these results
indicate that two complementary mechanisms combine to account for the
greater ability of high-avidity CTL to prevent an increase in viral
titer by killing infected cells before the assembly and release of new
virus: 1) high-avidity CTL recognize and kill virus-infected cells
earlier in the course of infection at very low Ag densities and 2)
high-avidity CTL have more rapid initiation of lysis and thus a more
rapid elimination of infected targets before substantial production and
release of functional virus. This latter mechanism may reflect more
efficient TCR signaling, as suggested by differences in functional
avidity in transgenic cells with the identical TCR, or in cells in
which the TCR affinity did not correlate completely with functional
avidity.4,5
Although both mechanisms may represent corollaries that derive from the
same fundamental property, they actually represent functionally
different aspects operating independently in time and complementing
each other to produce the greater efficacy of high-avidity CTL. One
corresponds to the generation of Ag density during virus replication,
whereas the other reflects the rate at which CTL are able to recognize
that Ag density during TCR signaling. Thus, in vivo, enhanced
recognition of low levels of viral Ag and superior TCR kinetics are
likely to be the determining factors in the differential ability of
high-avidity CTL to reduce viral titers.
The lag in onset of lysis by low- vs high-avidity CTL, or by high-avidity CTL at very low Ag densities, is of interest in its own right. One of the factors contributing to CTL avidity is the affinity of the TCR. One might speculate that the onset of lysis requires some threshold number of TCR molecules to be engaged or aggregated (39) or to be serially triggered (40, 41). In either case, since low-affinity TCRs would be expected to have a much shorter average dwell time for engagement of the peptide-MHC complex than that of high-affinity TCRs, the probability that an encounter between a TCR and its ligand would last long enough to result in a productive signal would be lower for low-avidity CTL. Therefore, it might take longer to achieve the threshold necessary for activation of the CTL. Similarly, even for high-affinity TCRs, if the density of relevant peptide-MHC on the target cell was too sparse, it might take longer to accumulate sufficient interactions to reach this threshold.
Our previous work demonstrated that adoptively transferred high-avidity
CTL could effectively prevent the accumulation of high vaccinia titers
in the ovary, the principle site of vaccinia proliferation in female
mice (Ref. 22 and data not shown), whereas low-avidity CTL
were generally ineffective. To explore other possible mechanisms that
might also contribute to reduction in viral load, in this work we
examine whether low-avidity CTL would be more effective either at
higher concentrations or after an extended period of interaction with
infected cells in vivo. This is particularly relevant, since, in vitro,
high-avidity CTL both recognize lower Ag density and initiate lysis
more rapidly than low-avidity CTL, and others have shown that time is
critical in an effective in vivo response (38). Fig. 5
demonstrates that high-avidity CTL are clearly more effective at
reducing viral titers than low-avidity CTL, but, interestingly, viral
titer increased rapidly with a small decrease in the number of
high-avidity CTL transferred. One explanation for this might be that we
used a SCID model system to examine adoptive transfer to isolate the
effects of the CD8+ CTL on the reduction of viral
titer. SCID mice lack those components of the immune response that
might be expected to effectively clear viral particles. Adoptively
transferred CTL can reduce the accumulation of new virus, but are
unlikely to reduce virus once already produced. Interestingly, when
Gallimore et al. (23) transferred CTL into immunologically
intact B6 mice, they also found that high-avidity CTL reduced viral
titers more than lower avidity CTL, but in their system protection by
both high- and low-avidity CTL showed substantial improvements as the
number of CTL transferred increased. Viral titers measured in the ovary
on day 3 or 6 were heavily influenced by the initial viral burden
injected on day 0. If the initial viral burden were sufficiently high,
it was impossible to demonstrate a reduction in viral titer on day 3 or
6, even following the transfer of 3 x 107
high-avidity CTL (data not shown). Thus, in this SCID model system,
adoptively transferred CTL must recognize infected cells early enough
in the course of viral replication that they prevent the accumulation
of new virus particles, but if the initial viral burden is too high,
their efforts may go unrecognized. Low-avidity CTL would then not be
expected to be more effective given increased time in contact with
infected cells, since even if they could recognize higher Ag densities
at later time points, high viral titers would already have accumulated.
The results in Fig. 2, showing that high-avidity CTL are capable of
recognizing infected target cells much earlier than low-avidity CTL, as
well as those in Fig. 4, showing that low-avidity CTL exhibit a delay
in the onset of lysis, argue that by the time low-avidity CTL could
recognize infected cells in vivo, viral progeny might already be
assembled. Consistent with this prediction, Fig. 6 shows that, after
adoptive transfer and infection of mice with vPE16, low-avidity CTL
were no more effective at reducing viral titers by day 6 than they had
been on day 3.
Yet another mechanism that we had to consider was the possibility
raised by several studies that differences in homing could affect the
ability of adoptively transferred lymphocytes to function properly in
vivo (16, 42, 43). CD44 in particular has been reported to
determine whether adoptively transferred clones were capable of
preventing malaria as a result of infection with Plasmodium
yoelii (44). The CTL lines and clones in our
experiments do not show differences in the expression of CD44 that
correlated with their avidity (data not shown). In addition, the
enhanced ability of high-avidity CTL to reduce viral titers following
adoptive transfer into infected SCID mice has been observed in three
independently generated sets of lines as well as with CTL clones
developed from those lines. To formally address the question of
differences in the homing of high- and low-avidity CTL, however, CTL
were labeled with fluorescent markers and transferred into SCID mice
simultaneously infected with recombinant vaccinia. Ovaries harvested at
24 and 48 h showed no difference in the presence of labeled high-
or low-avidity CTL in the ovary (Fig. 7), and, thus, no difference in
the ability of either high- or low-avidity CTL to home to the site of
infection, a result consistent with that seen in another model system
(45).
In conclusion, two complementary mechanisms acting together appear to account for the greater efficacy of high-avidity CTL. First, high-avidity CTL recognize lower Ag densities present earlier in the course of infection of each cell, and, second, they initiate a more rapid lysis than low-avidity CTL. Both mechanisms allow them to eliminate cells infected with virus earlier. Early recognition and elimination of infected cells prevents the accumulation of virus and limits the extent of the resulting infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jay A. Berzofsky, Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1578.
3 Abbreviations used in this paper: diIC16, 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; BATDA, bis(acetoxymethyl)-2,2':6',2'-terpyridine-6,6'-dicarboxylate.
4 M. A. Derby, J. Wang, D. H. Margulies, and J. A. Berzofsky. Two intermediate avidity CTL clones with a disparity between functional avidity and MHC tetramer staining. Submitted for publication.
5 A. G. Cawthon, H. Lu, and M. A. Alexander-Miller. Sensitivity to TCR engagement as a mechanism to control CTL avidity. Submitted for publication.
Received for publication July 19, 2000. Accepted for publication November 14, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  M. Terabe, E. Ambrosino, S. Takaku, J. J. O'Konek, D. Venzon, S. Lonning, J. M. McPherson, and J. A. Berzofsky Synergistic Enhancement of CD8+ T Cell-Mediated Tumor Vaccine Efficacy by an Anti-Transforming Growth Factor-{beta} Monoclonal Antibody Clin. Cancer Res., November 1, 2009; 15(21): 6560 - 6569. [Abstract] [Full Text] [PDF]
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 J. R. Almeida, D. Sauce, D. A. Price, L. Papagno, S. Y. Shin, A. Moris, M. Larsen, G. Pancino, D. C. Douek, B. Autran, et al. Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity Blood, June 18, 2009; 113(25): 6351 - 6360. [Abstract] [Full Text] [PDF]
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 B. C. Simons, S. E. VanCompernolle, R. M. Smith, J. Wei, L. Barnett, S. L. Lorey, D. Meyer-Olson, and S. A. Kalams Despite Biased TRBV Gene Usage against a Dominant HLA B57-Restricted Epitope, TCR Diversity Can Provide Recognition of Circulating Epitope Variants J. Immunol., October 1, 2008; 181(7): 5137 - 5146. [Abstract] [Full Text] [PDF]
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Z. Sebestyen, E. Schooten, T. Sals, I. Zaldivar, E. San Jose, B. Alarcon, S. Bobisse, A. Rosato, J. Szollosi, J. W. Gratama, et al. Human TCR That Incorporate CD3{zeta} Induce Highly Preferred Pairing between TCR{alpha} and {beta} Chains following Gene Transfer J. Immunol., June 1, 2008; 180(11): 7736 - 7746. [Abstract] [Full Text] [PDF]
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 N. P. Andrews, C. D. Pack, and A. E. Lukacher Generation of Antiviral Major Histocompatibility Complex Class I-Restricted T Cells in the Absence of CD8 Coreceptors J. Virol., May 15, 2008; 82(10): 4697 - 4705. [Abstract] [Full Text] [PDF]
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G. Plesa, A. E. Snook, S. A. Waldman, and L. C. Eisenlohr Derivation and Fluidity of Acutely Induced Dysfunctional CD8+ T Cells J. Immunol., April 15, 2008; 180(8): 5300 - 5308. [Abstract] [Full Text] [PDF]
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D. Yerly, D. Heckerman, T. M. Allen, J. V. Chisholm III, K. Faircloth, C. H. Linde, N. Frahm, J. Timm, W. J. Pichler, A. Cerny, et al. Increased Cytotoxic T-Lymphocyte Epitope Variant Cross-Recognition and Functional Avidity Are Associated with Hepatitis C Virus Clearance J. Virol., March 15, 2008; 82(6): 3147 - 3153. [Abstract] [Full Text] [PDF]
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 I. M. Belyakov, S. Kozlowski, M. Mage, J. D. Ahlers, L. F. Boyd, D. H. Margulies, and J. A. Berzofsky Role of {alpha}3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs Int. Immunol., December 1, 2007; 19(12): 1413 - 1420. [Abstract] [Full Text] [PDF]
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Y. Liu, J. McNevin, H. Zhao, D. M. Tebit, R. M. Troyer, M. McSweyn, A. K. Ghosh, D. Shriner, E. J. Arts, M. J. McElrath, et al. Evolution of Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitopes: Fitness-Balanced Escape J. Virol., November 15, 2007; 81(22): 12179 - 12188. [Abstract] [Full Text] [PDF]
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 J. R. Almeida, D. A. Price, L. Papagno, Z. A. Arkoub, D. Sauce, E. Bornstein, T. E. Asher, A. Samri, A. Schnuriger, I. Theodorou, et al. Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover J. Exp. Med., October 1, 2007; 204(10): 2473 - 2485. [Abstract] [Full Text] [PDF]
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M. S. Bennett, H. L. Ng, M. Dagarag, A. Ali, and O. O. Yang Epitope-Dependent Avidity Thresholds for Cytotoxic T-Lymphocyte Clearance of Virus-Infected Cells J. Virol., May 15, 2007; 81(10): 4973 - 4980. [Abstract] [Full Text] [PDF]
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M. Lichterfeld, X. G. Yu, S. K. Mui, K. L. Williams, A. Trocha, M. A. Brockman, R. L. Allgaier, M. T. Waring, T. Koibuchi, M. N. Johnston, et al. Selective Depletion of High-Avidity Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD8+ T Cells after Early HIV-1 Infection J. Virol., April 15, 2007; 81(8): 4199 - 4214. [Abstract] [Full Text] [PDF]
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A. H. Dzutsev, I. M. Belyakov, D. V. Isakov, D. H. Margulies, and J. A. Berzofsky Avidity of CD8 T cells sharpens immunodominance Int. Immunol., April 1, 2007; 19(4): 497 - 507. [Abstract] [Full Text] [PDF]
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C. Ranasinghe, S. J. Turner, C. McArthur, D. B. Sutherland, J.-H. Kim, P. C. Doherty, and I. A. Ramshaw Mucosal HIV-1 Pox Virus Prime-Boost Immunization Induces High-Avidity CD8+ T Cells with Regime-Dependent Cytokine/Granzyme B Profiles J. Immunol., February 15, 2007; 178(4): 2370 - 2379. [Abstract] [Full Text] [PDF]
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R. H. Mealey, J.-H. Lee, S. R. Leib, M. H. Littke, and T. C. McGuire A Single Amino Acid Difference within the {alpha}-2 Domain of Two Naturally Occurring Equine MHC Class I Molecules Alters the Recognition of Gag and Rev Epitopes by Equine Infectious Anemia Virus-Specific CTL J. Immunol., November 15, 2006; 177(10): 7377 - 7390. [Abstract] [Full Text] [PDF]
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H. Horton, C. Havenar-Daughton, D. Lee, E. Moore, J. Cao, J. McNevin, T. Andrus, H. Zhu, A. Rubin, T. Zhu, et al. Induction of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses in HIV Vaccine Trial Participants Who Subsequently Acquire HIV-1 Infection J. Virol., October 1, 2006; 80(19): 9779 - 9788. [Abstract] [Full Text] [PDF]
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A. C. M. Boon, G. de Mutsert, R. A. M. Fouchier, A. D. M. E. Osterhaus, and G. F. Rimmelzwaan The Hypervariable Immunodominant NP418-426 Epitope from the Influenza A Virus Nucleoprotein Is Recognized by Cytotoxic T Lymphocytes with High Functional Avidity. J. Virol., June 1, 2006; 80(12): 6024 - 6032. [Abstract] [Full Text] [PDF]
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I. M. Belyakov, V. A. Kuznetsov, B. Kelsall, D. Klinman, M. Moniuszko, M. Lemon, P. D. Markham, R. Pal, J. D. Clements, M. G. Lewis, et al. Impact of vaccine-induced mucosal high-avidity CD8+CTLs in delay of AIDS viral dissemination from mucosa Blood, April 15, 2006; 107(8): 3258 - 3264. [Abstract] [Full Text] [PDF]
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 L. T. van den Broeke, C. D. Pendleton, C. Mackall, L. J. Helman, and J. A. Berzofsky Identification and Epitope Enhancement of a PAX-FKHR Fusion Protein Breakpoint Epitope in Alveolar Rhabdomyosarcoma Cells Created by a Tumorigenic Chromosomal Translocation Inducing CTL Capable of Lysing Human Tumors Cancer Res., February 1, 2006; 66(3): 1818 - 1823. [Abstract] [Full Text] [PDF]
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D. A. Price, J. M. Brenchley, L. E. Ruff, M. R. Betts, B. J. Hill, M. Roederer, R. A. Koup, S. A. Migueles, E. Gostick, L. Wooldridge, et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses J. Exp. Med., November 21, 2005; 202(10): 1349 - 1361. [Abstract] [Full Text] [PDF]
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 V. Lennerz, M. Fatho, C. Gentilini, R. A. Frye, A. Lifke, D. Ferel, C. Wolfel, C. Huber, and T. Wolfel The response of autologous T cells to a human melanoma is dominated by mutated neoantigens PNAS, November 1, 2005; 102(44): 16013 - 16018. [Abstract] [Full Text] [PDF]
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S. Yang, J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL J. Immunol., September 15, 2005; 175(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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P. M. Lavoie, A. R. Dumont, H. McGrath, A.-E. Kernaleguen, and R.-P. Sekaly Delayed expansion of a restricted T cell repertoire by low-density TCR ligands Int. Immunol., July 1, 2005; 17(7): 931 - 941. [Abstract] [Full Text] [PDF]
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J. W. Hodge, M. Chakraborty, C. Kudo-Saito, C. T. Garnett, and J. Schlom Multiple Costimulatory Modalities Enhance CTL Avidity J. Immunol., May 15, 2005; 174(10): 5994 - 6004. [Abstract] [Full Text] [PDF]
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S. Oh, L. P. Perera, D. S. Burke, T. A. Waldmann, and J. A. Berzofsky IL-15/IL-15R{alpha}-mediated avidity maturation of memory CD8+ T cells PNAS, October 19, 2004; 101(42): 15154 - 15159. [Abstract] [Full Text] [PDF]
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 G. R. Leggatt, S. Narayan, G. J. P. Fernando, and I. H. Frazer Changes to peptide structure, not concentration, contribute to expansion of the lowest avidity cytotoxic T lymphocytes J. Leukoc. Biol., October 1, 2004; 76(4): 787 - 795. [Abstract] [Full Text] [PDF]
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M. Regner, X. Martinez, E. Belnoue, C.-M. Sun, F. Boisgerault, P.-H. Lambert, C. Leclerc, and C.-A. Siegrist Partial Activation of Neonatal CD11c+ Dendritic Cells and Induction of Adult-Like CD8+ Cytotoxic T Cell Responses by Synthetic Microspheres J. Immunol., August 15, 2004; 173(4): 2669 - 2674. [Abstract] [Full Text] [PDF]
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 H. Reijonen, R. Mallone, A.-K. Heninger, E. M. Laughlin, S. A. Kochik, B. Falk, W. W. Kwok, C. Greenbaum, and G. T. Nepom GAD65-Specific CD4+ T-Cells with High Antigen Avidity Are Prevalent in Peripheral Blood of Patients With Type 1 Diabetes Diabetes, August 1, 2004; 53(8): 1987 - 1994. [Abstract] [Full Text] [PDF]
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R. Draenert, C. L. Verrill, Y. Tang, T. M. Allen, A. G. Wurcel, M. Boczanowski, A. Lechner, A. Y. Kim, T. Suscovich, N. V. Brown, et al. Persistent Recognition of Autologous Virus by High-Avidity CD8 T Cells in Chronic, Progressive Human Immunodeficiency Virus Type 1 Infection J. Virol., January 15, 2004; 78(2): 630 - 641. [Abstract] [Full Text] [PDF]
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C. L. Meier, M. Svensson, and P. M. Kaye Leishmania-Induced Inhibition of Macrophage Antigen Presentation Analyzed at the Single-Cell Level J. Immunol., December 15, 2003; 171(12): 6706 - 6713. [Abstract] [Full Text] [PDF]
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J. Cao, J. McNevin, U. Malhotra, and M. J. McElrath Evolution of CD8+ T Cell Immunity and Viral Escape Following Acute HIV-1 Infection J. Immunol., October 1, 2003; 171(7): 3837 - 3846. [Abstract] [Full Text] [PDF]
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T. W. Kim, C.-F. Hung, D. Boyd, J. Juang, L. He, J. W. Kim, J. M. Hardwick, and T.-C. Wu Enhancing DNA Vaccine Potency by Combining a Strategy to Prolong Dendritic Cell Life with Intracellular Targeting Strategies J. Immunol., September 15, 2003; 171(6): 2970 - 2976. [Abstract] [Full Text] [PDF]
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T. C. McGuire, S. R. Leib, R. H. Mealey, D. G. Fraser, and D. J. Prieur Presentation and Binding Affinity of Equine Infectious Anemia Virus CTL Envelope and Matrix Protein Epitopes by an Expressed Equine Classical MHC Class I Molecule J. Immunol., August 15, 2003; 171(4): 1984 - 1993. [Abstract] [Full Text] [PDF]
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S. Oh, J. W. Hodge, J. D. Ahlers, D. S. Burke, J. Schlom, and J. A. Berzofsky Selective Induction of High Avidity CTL by Altering the Balance of Signals from APC J. Immunol., March 1, 2003; 170(5): 2523 - 2530. [Abstract] [Full Text] [PDF]
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P. M. Gray, G. D. Parks, and M. A. Alexander-Miller High Avidity CD8+ T Cells Are the Initial Population Elicited Following Viral Infection of the Respiratory Tract J. Immunol., January 1, 2003; 170(1): 174 - 181. [Abstract] [Full Text] [PDF]
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T. U. Vogel, T. C. Friedrich, D. H. O'Connor, W. Rehrauer, E. J. Dodds, H. Hickman, W. Hildebrand, J. Sidney, A. Sette, A. Hughes, et al. Escape in One of Two Cytotoxic T-Lymphocyte Epitopes Bound by a High-Frequency Major Histocompatibility Complex Class I Molecule, Mamu-A*02: a Paradigm for Virus Evolution and Persistence? J. Virol., October 11, 2002; 76(22): 11623 - 11636. [Abstract] [Full Text] [PDF]
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P. Chames, R. A. Willemsen, G. Rojas, D. Dieckmann, L. Rem, G. Schuler, R. L. Bolhuis, and H. R. Hoogenboom TCR-Like Human Antibodies Expressed on Human CTLs Mediate Antibody Affinity-Dependent Cytolytic Activity J. Immunol., July 15, 2002; 169(2): 1110 - 1118. [Abstract] [Full Text] [PDF]
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 G. D. Parks and M. A. Alexander-Miller High avidity cytotoxic T lymphocytes to a foreign antigen are efficiently activated following immunization with a recombinant paramyxovirus, simian virus 5 J. Gen. Virol., May 1, 2002; 83(5): 1167 - 1172. [Abstract] [Full Text] [PDF]
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U. Kumaraguru, M. Gierynska, S. Norman, B. D. Bruce, and B. T. Rouse Immunization with Chaperone-Peptide Complex Induces Low-Avidity Cytotoxic T Lymphocytes Providing Transient Protection against Herpes Simplex Virus Infection J. Virol., January 1, 2002; 76(1): 136 - 141. [Abstract] [Full Text] [PDF]
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M. J. Estcourt, A. J. Ramsay, A. Brooks, S. A. Thomson, C. J. Medveckzy, and I. A. Ramshaw Prime-boost immunization generates a high frequency, high-avidity CD8+ cytotoxic T lymphocyte population Int. Immunol., January 1, 2002; 14(1): 31 - 37. [Abstract] [Full Text] [PDF]
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P. M. Gray, G. D. Parks, and M. A. Alexander-Miller A Novel CD8-Independent High-Avidity Cytotoxic T-Lymphocyte Response Directed against an Epitope in the Phosphoprotein of the Paramyxovirus Simian Virus 5 J. Virol., November 1, 2001; 75(21): 10065 - 10072. [Abstract] [Full Text] [PDF]
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