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*
Department of Medicine, University of Western Australia, Nedlands, Australia; and
West Australian Institute for Medical Research, Queen Elizabeth II Medical Center, Perth, Australia
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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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Presentation of Ags via the classical class I pathway has demonstrated that CTL responses are often limited to a few, or even a single, dominant peptides, which, in some instances, may be associated with weaker responses to other epitopes, termed subdominant (reviewed in Ref. 13). Responses to subdominant epitopes have been shown to be biologically significant. In particular, studies examining viral infections have demonstrated T cell responses to epitopes of varying affinities (14, 15). Similarly, autoimmune models have demonstrated the sequential revealing of T cell responses to weaker epitopes during disease (16, 17). In some tumor systems, CTL to subdominant peptides have been detected during tumor growth (18) and, when these peptides are used as immunogens, they are able to confer protection (19). However, studies looking at hierarchical CTL responses to soluble proteins have generally been limited to in vitro systems (20, 21), and no studies to date have addressed Ag cross-presentation and its relationship to the in vivo generation and subsequent functional activity of CTL specific for subdominant or cryptic peptides.
In this study, the in vivo relationship between local Ag cross-presentation and effector CTL responses after a single injection vs chronic exposure to a soluble Ag was assessed. Following a single exposure of OVA, Ag cross-presentation was maximal in, but not restricted to, the draining lymph nodes (DLN)3 and continued for 3 wk, a process much more prolonged than previously considered. Fully functional in vivo effector CTL recognizing the dominant peptide were readily detectable within, but not after, the period spanning Ag presentation, suggesting that loss of antigenic stimulation is directly associated with loss of effector function. The presence of IFA not only prolonged Ag presentation, promoted Ag presentation to low antigenic concentrations, and generated qualitative changes in functional effector CTL, but also revealed T cell precursor responses to a subdominant epitope. This work showed that induction of T cell responses to weaker epitopes requires continual cross-presentation of high antigenic concentrations within an adjuvant/inflammatory milieu. However, subdominant-specific CTL activity could not be seen in vivo, possibly due to the presence of a concomitant CTL response to the dominant epitope.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6J mice (H-2b) were obtained from the Animal Resources Center (Canning Vale, Western Australia) and maintained under standard housing conditions in the University Department of Medicine (University of Western Australia) animal holding area. The TCR-transgenic mouse line OT-1, expressing a TCR recognizing an H-2b-restricted OVA epitope, SIINFEKL (22), was kindly supplied by F. Carbone (Monash University, Melbourne, Australia). For all experiments, mice between the ages of 6 and 12 wk were used.
OVA and peptides
OVA, grade V and grade VI (Sigma, St. Louis, MO), the model protein Ag used throughout these experiments, was regularly screened using a proliferation assay to ensure that the protein had not broken down to release the SIINFEKL peptide during routine handling procedures.
The dominant peptide OVA275–264 (SIINFEKL), a subdominant peptide OVA55–62, KVVRFDKL (21, 23), and a cryptic peptide OVA11–18, CFDVFKEL (20), were manufactured by the Center for Cell and Molecular Biology (University of Western Australia, Perth) at purities of 89, 90, and 95%, respectively.
Proliferation assay
Spleen cells from unmanipulated mice were irradiated (2100 rad), fixed with 4% formaldehyde (10 min at room temperature), washed twice, and pulsed with varying concentrations of OVA or SIINFEKL (90 min at 37°C under 5% CO2). Washed APC were plated in triplicate, with 2 x 105 OT-1 lymph node cells in 96-well flat-bottom microtiter plates (Costar, Cambridge, MA), in 200 µl of complete medium (CM). CM consisted of RPMI 1640 supplemented with 10% FCS (both from Life Technologies, Gaithersburg, MD) containing 100 U/ml penicillin (CSL, Melbourne, Australia), 50 µg/ml gentamicin (David Bull Laboratories, Mulgrave North, Australia), and 5 x 10-5 M 2-ME (Sigma). The cells were incubated for 48 and 72 h and 1 µCi/well [3H]thymidine (Amersham, U.K.) was added 12–15 h before harvesting. In all tests, OVA grade V was found to remain free of peptide; however, on occasions, OVA grade VI did have peptide contamination and was therefore not used in our experimental procedures.
Vaccination protocol
Mice were given varying doses of OVA either dissolved in PBS or as an emulsion in IFA. Mice were s.c. injected with 100 µl at two sites on either side of the base of the tail.
Cytoplasmic loading of OVA into APC using osmotic shock
Osmotic loading was conducted as previously described (24). Briefly, after erythrocytes were removed by lysis, C57BL/6 spleen cell suspensions were washed and pulsed with 1 mg/ml OVA in 1 ml of hypertonic media (0.5 M sucrose, 10% (w/v) polyethylene glycol, 10 mM HEPES, in RPMI 1640 at pH 7.2) for 10 min at 37°C. Then, 14 ml of hypotonic medium (60% RPMI 1640 and 40% H2O) was added for 2 min at 37°C, followed by centrifugation at 1200 rpm for 5 min, washed twice, and placed into culture.
Murine tumor cell lines
The EL4 thymoma cell line and the OVA-transfected EL4 cell line EG7 (24) were both purchased from the American Type Culture Collection (Manassas, VA) and maintained in CM.
5,6-Carboxy-fluorescein succinimdyl ester (CFSE) labeling of OT-1 TCR-transgenic lymph node cells
CFSE (Molecular Probes, Eugene, OR) labeling was performed as previously described (25). SIINFEKL-specific lymph node cells from TCR-transgenic OT-1 mice were resuspended in 20 ml of RPMI 1640 at 1 x 107 cells/ml and incubated with 1 µl of CFSE stock solution (5 mM in DMSO) for 10 min at room temperature. Cells were washed through FCS four times and PBS twice, then resuspended in PBS before 1 x 107 cells were i.v. injected into the tail vein of each recipient mouse. In all experiments, CFSE-labeled cells were recovered 3 days after adoptive transfer and analyzed by FACS analysis. CFSE-labeled cells were adoptively transferred at time points such that the degree of proliferation was analyzed on days 7, 14, 21, and 28 after OVA immunization. In a few instances, other time points were also examined (as described in Results and the figure legends).
FACS analysis
Lymph node or spleen cells were stained for two-color FACS analysis using the PE-conjugated mAb anti CD8-(clone 53-6.7; PharMingen, San Diego, CA). Analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA) using CellQuest software. For analysis of CFSE-labeled cells, 100,000 events were collected and analyzed using the ModFit LT cell cycle analysis software (Verity Software House, Topsham, ME).
In vitro cytotoxic assay
Spleens were removed and prepared as single-cell suspensions. To ensure that there were enough effector CTL for the assay, cells from each experimental group (of three mice) were pooled. These effector cells were then expanded in vitro with a 1:1 ratio of splenic APC taken from naive mice that were either osmotically loaded with whole native OVA or pulsed for 90 min with 10-6 M of the dominant, subdominant, or cryptic peptides. All populations of APC were thoroughly washed before addition to the effector cells taken from experimental mice and incubated at 37°C for 5 days in CM.
Target cells, which included the OVA transfectant cell line EG7, and EL4 cells pulsed with either SIINFEKL, KVVRFDKL, or the cryptic peptide were labeled with 150 µCi of 51Cr for 90 min and washed four times before use. Effector cells were added to corresponding targets at varying E:T cell ratios and incubated at 37°C for 4 h. After incubation, the supernatants were harvested and 51Cr release was determined. The mean of duplicate samples was calculated and the percent specific 51Cr release was determined as follows: percentage of specific cytolysis = [(experimental 51Cr release - control 51Cr release)/(maximum 51Cr release - control Cr51 release)] x 100%. Experimental 51Cr release represents counts from target cells mixed with effector cells, control 51Cr release represents counts from targets incubated with medium alone (spontaneous release), and maximum 51Cr release represents counts from targets exposed to 5% Triton X-100.
In vivo cytotoxicity assay
Target cells for in vivo evaluation of cytotoxic activity were prepared as described elsewhere (26). Briefly, erythrocytes from C57BL/6 pooled spleen and lymph node cell suspensions were lysed, and the cells were washed and then divided into two or three populations. One population was pulsed with 10-6 M SIINFEKL, and, where stated, a second population was pulsed with 10-6 M KVVRFDKL for 90 min at 37°C, washed in PBS, and labeled with high (5 µM) or low (0.05 µM) CFSE concentrations, respectively. Control uncoated target cells were labeled with an intermediate concentration of CFSE (0.5 µM). For i.v. injection, 1 x 107 cells of each population was mixed in 200 µl of PBS per recipient mouse. Specific in vivo cytotoxicity was determined by collecting the DLN, non-DLN, and spleen from recipient mice 18 h after i.v. injection and differentially labeled fluorescent target cell populations detected by flow cytometry. The ratio between the percentages of uncoated vs SIINFEKL-coated (CFSEint/CFSEhigh) was calculated to obtain a numerical value of cytotoxicity. Further controls included naive, PBS-, or IFA-only-treated recipient mice. To normalize data allowing interexperimental comparisons, ratios were calculated between the percentages of peptide coated in control vs OVA-exposed mice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first series of experiments was designed to dissect out the relationship between Ag concentration and duration of Ag presentation (in this case, the dominant class I peptide, SIINFEKL) in the absence of an adjuvant. A kinetic study was undertaken in which CFSE-labeled OT-1 lymph node cells were adoptively transferred into recipient mice treated with a single s.c. injection of varying concentrations of OVA diluted in PBS. Throughout these experiments, the proliferative responses of CFSE-labeled OT-1 cells in secondary lymphoid compartments of recipient animals were analyzed 3 days after adoptive transfer. Responses were determined weekly over 1 mo.
A strong in vivo OT-1 proliferative response was seen in the DLN of all
mice given 200 µg of OVA (Fig. 1
). This
response continued for 2 wk, diminished at day 21, and disappeared by
day 28. In mice given an intermediate dose (20 µg OVA), only one of
three mice demonstrated a weak response 7 days after OVA immunization,
and no response was observed at any other time point. No OT-1
proliferation was seen in the DLN at any time point in mice given the
lowest concentration (2 µg of OVA).
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compares the magnitude of the
proliferative response determined by quantifying the percentage of
daughter cells relative to those remaining in the parental peak in
different lymphoid tissues. The highest concentration of OVA induced a
strong proliferative response in the DLN (65% of cells had undergone
at least one division), in association with significant but lower
levels of proliferation in the nondraining LN (29% daughter cells),
and an even weaker, but positive response in the spleen (11%
proliferating cells). Log-fold dilutions of OVA induced correspondingly
lower levels of SIINFEKL-specific OT-1 proliferation at day 7 in all of
the secondary lymphoid organs examined. Unlike the highest OVA dose,
variable OT-1 proliferative responses were seen in the two lower doses
at days 14 and 21 after inoculation. Interestingly, all doses at days 7
and 21 after immunization showed weak, but above background levels of
proliferation within the spleen, implying low levels of Ag
presentation. However, by day 28, no proliferation was seen in any
tissue site. These data imply that high doses of soluble protein are
required before consistent Ag presentation can be observed and that, at
this concentration, cross-presentation is observed not only within the
DLN, but also throughout the secondary lymphoid system.
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Based on the above observations, we assessed whether peptide-specific effector CTL cells had been generated in mice given the same doses of OVA using an in vitro 51Cr release CTL assay. In this assay, spleens were pooled from each experimental group (of three mice) and expanded in vitro with splenic APC taken from naive mice. These APC were either osmotically loaded with whole native OVA or pulsed with the dominant peptide.
At all time points examined (Fig. 2), the
highest concentration of OVA yielded the strongest CTL response to the
dominant peptide. However, in contrast to Ag presentation within the
DLN, this response was maintained well beyond 28 days and remained
readily detectable 8 wk later, declining to a weak, but detectable CTL
response at 3 mo after immunization. There appears to be some
interexperimental variation with responses to the lower OVA
concentrations. For example, at day 7, a higher CTL response was seen
to the 2-µg dose than to the 20-µg dose. Days 28 and 56 were more
consistent, with the two lower OVA doses demonstrating equivalent CTL
responses.
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shows the percent lysis by effector cells cultured in vitro with
SIINFEKL-pulsed APC and tested on SIINFEKL-pulsed EL4 targets. However,
similar data were generated when the effector cells were pulsed with
osmotically loaded APC (data not shown), as well as when the
OVA-transfected cell line EG7 was used as a target (data not shown).
These data demonstrated that SIINFEKL-specific CTL were detectable in
spleens taken from experimental mice that had received all three OVA
doses. In vivo effector CTL are present throughout the lymphoid system
The use of in vitro antigenic re-exposure to expand
peptide-specific CTL precursors (seen in Fig. 2) may not be an accurate
reflection of in vivo physiological events. Therefore, an in vivo CTL
assay was used to assess the location and duration of effector CTL in
mice immunized with 200 µg of OVA in PBS. In this assay,
differentially CFSE-labeled target cells from naive mice were pulsed
with (CFSEhigh) or without SIINFEKL
(CFSEint), i.v. injected into experimental mice
and CTL responses within secondary lymphoid organs examined 18 h
later.
A strong in vivo SIINFEKL-specific effector CTL response (illustrated
in an individual mouse in Fig. 3 and as
group means in Fig. 4) was seen in the
DLN in association with a weaker response in the spleen at day 7.
Variable splenic responses were seen, with one mouse showing equivalent
effector responses throughout the secondary lymphoid system (Fig. 3)
and others demonstrating weaker responses (Fig. 4). We were surprised
to see strong CTL activity throughout the secondary lymphoid system at
day 7. However, unlike the in vitro detection system, in vivo
peptide-specific CTL responses rapidly declined to barely detectable
levels by day 20.
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Prolonged exposure to Ag extends the period of Ag presentation and enhances responses to lower doses
Peptides, proteins, heat-killed intact cells, or cell lysates in
the presence of adjuvants such as IFA are used for a number of
vaccination purposes, including tumor immunotherapy. Based on the above
results, we evaluated how responses to OVA in the presence of IFA
altered the immune response. As IFA acts as an oil/emulsion Ag depot,
we were not surprised to find that, at the highest OVA concentration
used (200 µg), Ag presentation within the DLN persisted well past 28
days (Fig. 5), diminishing to below
detectable levels 8 wk after inoculation (data not shown). However, the
presence of IFA also promoted increased sensitivity to lower antigenic
concentrations. Immunization with 20 µg of OVA in IFA also resulted
in persistent Ag presentation throughout the time period examined (28
days). Weak levels of Ag presentation were detected 7 days after
exposure to 2 µg and 0.2 µg (data not shown), but not at any other
time. No Ag presentation was seen when mice were inoculated with 0.02
µg of OVA in IFA (data not shown).
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Effector/memory CTL responses to SIINFEKL, determined by a
51Cr release in vitro CTL assay, demonstrated
equivalent responses to the two higher OVA concentrations in IFA for up
to 2 wk after inoculation (Fig. 6). These
responses became dose responsive 3 wk after exposure and persisted past
28 days. Peptide-specific CTL responses generated by exposure to the
lowest dose demonstrated variable results between experiments;
nevertheless, they were significantly reduced at the earlier time
points and diminished to background levels 3 wk later.
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Comparison of the in vivo CTL response following administration of
200 µg of OVA in IFA to those seen after OVA alone treatment (Fig. 7) showed that IFA only marginally
increased the level of peptide-specific CTL in the DLN and LN at day 7.
However, the splenic response was almost doubled (to 65% lysis) at
this time point. These responses diminished to relatively constant
levels for the duration of the experiment. In contrast to Ag exposure
in the absence of IFA, effector activity within the DLN consistently
demonstrated higher effector CTL activity throughout the
experiment.
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We evaluated the generation of CTLs with specificity for a defined
subdominant peptide using the standard 51Cr
release CTL assay in which effector cells were expanded in vitro with
the subdominant peptide, and target cells were pulsed with the same
peptide. Fig. 8 shows that a sustained
subdominant response was detected in spleens taken from mice given 200
µg or 20 µg, but not to mice given 2 µg of OVA in IFA. A weak and
transient subdominant-specific CTL response was also seen in mice given
200 µg of OVA only. This CTL response to the subdominant peptide was
quantitatively weaker than those seen to the dominant peptide within
the same groups of mice (Fig. 8 vs Fig. 6), suggesting the generation
of lower, but not insignificant numbers of subdominant vs dominant
precursor CTL. Additionally, effector cells expanded in vitro with the
cryptic peptide did not demonstrate lytic activity toward targets
pulsed with the cryptic peptide (data not shown), indicating that CTL
specific to this epitope were not generated. Hence, the generation of
responses to a subdominant peptide requires cross-presentation from a
persistent and highly concentrated source of Ag.
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) or other secondary lymphoid sites
(data not shown) examined at any time point. Taken together, these data
suggest that, although CTL to the subdominant peptide are generated in
mice given high OVA doses plus IFA, in vivo these CTL are poor
effectors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dose, distribution, and duration of cross-presented protein Ag
Until now, the relationship between dose and location of Ag
presentation of noninfectious protein Ag in lymphoid tissues following
a single inoculation was unknown. When the threshold antigenic
concentration required for in vivo cross-presentation was examined, we
were surprised to find that a single inoculation of a high dose of OVA,
without adjuvant, results in Ag presentation that is detectable in the
DLN for up to 3 wk. Similarly, Kurts et al. (7) have shown
that cross-presentation of protein from an intact organ continued in
the DLN for 
10 days following removal of the organ. We believe that
the cell responsible for cross-presenting OVA (the most probable
candidate being the dendritic cell (DC); Refs. 8, 27)
remained in the DLN and, after 3 wk, declined to below detectable
levels for Ag presentation. This is supported by Garcia et al.
(28) who reported that DC presenting a specific Ag to CD4
T cells remained within the spleen for 3 wk before disappearing,
possibly via postmaturation, programmed DC apoptosis (29)
or T cell-mediated DC lysis (30).
Interestingly, very high doses also generated weaker, but significant TCR T cell proliferation in other lymph nodes and in the spleen. We have recently shown that high viral loads, as well as varying levels of tumor Ags, generate significant levels of TCR T cell proliferation that are restricted to the DLN (9, 10); therefore, we believe that proliferation in other lymphoid sites represents local Ag presentation and not migrating T cells. How the Ag was transported to these distal sites remains unknown. Soluble protein may have "leaked" into the blood supply or lymphatics, alternatively the protein may have been transported in association with a cell. It is also unclear whether the Ag was transported directly from the inoculation site or via the DLN. Promoting Ag persistence at the injection site (via IFA) not only prolonged cross-presentation (to 8 wk) but also enhanced sensitivity to lower OVA concentrations. This may be due to an increase in local DC numbers in response to inflammatory signals, as described in other models (31).
The relationship between cross-presentation and generation of CTL recognizing dominant vs subdominant epitopes
To date, the bulk of work addressing hierarchical responses to class I epitopes has been undertaken using infectious models, in particular viral systems (3, 4). Research on responses to weaker epitopes of soluble proteins has been limited to in vitro systems or artificial in vivo systems (e.g., using proteins forced into the cytosol; Ref. 32). We found that in vivo effector CTL activity specific for the dominant epitope reflected Ag concentration and directly paralleled the kinetics of Ag presentation. In vitro assays showed that dominant-specific CTL precursors could be detected beyond the point at which Ag presentation had ceased. However, we were unable to generate long-term memory CTL after exposure to OVA minus IFA, a process believed to be under the control of IL-2 and Ag concentration (Refs. 33, 34 and reviewed in Ref. 35).
Even in the absence of detectable Ag presentation, low antigenic concentrations resulted in the generation of dominant epitope-specific CTL, which may reflect 1) the ability of T cells to recognize vanishingly small numbers of peptide-class I complexes (36); 2) that we did not reach threshold detection levels; or 3) the Ag was rapidly processed, presented, and cleared within 7 days, implying a highly efficient system. In physiological terms, these data suggest that cross-priming Ag from infected cells requires small antigenic concentrations to induce an immune response and that the higher the dose the stronger the CTL response will be. It also implies that when local Ag presentation can be demonstrated in the absence of CTL, as reported in tumor models (37), there is a defect in CTL generation/maintenance rather than a failure of Ag cross-presentation to reach a threshold required for CTL generation. However, we have also shown that the context of Ag delivery may be crucial when a strong and persisting CTL is the desired outcome. Prolonging Ag presentation not only generated CTL responses, but also promoted increased CTL responses to lower antigenic doses. Hence, when low doses of OVA are used in the absence of IFA, a few DC may transport and present Ag to a limited number of T cells; however, IFA may recruit larger numbers of DC that, once in the DLN, reach threshold detection levels. Furthermore, as more local T cells are activated, the resulting T-DC interaction may promote DC survival (29), leading to sustained generation of effector CTL. This observation may be valuable when designing immunotherapeutic vaccines for use in diseases such as cancer, where large amounts of tumor Ag may be difficult to obtain. Thus, use of low concentrations of tumor Ag with an adjuvant that prolongs Ag presentation may be more likely to promote a promising clinical outcome.
A weak CTL response to the subdominant peptide was seen, after in vitro restimulation, in spleens taken from mice given the highest OVA concentration. However, changing the context of Ag presentation (and possibly Ag processing) to a persistent source revealed a dose responsive subdominant response. IFA may generate a local site of inflammation that is accompanied by a cellular infiltrate or enhance the APC capacity of cells already identified as either presenting a wider range of Ags or preferentially presenting weaker Ags, such as B cells (38) or fibroblasts (39), respectively. Analysis via a 51Cr release in vitro assay demonstrated that the subdominant epitope had been processed and presented, resulting in the generation of a significant number of specific precursor CTL. These observations may help explain the high numbers of dominant epitope-specific CTL generated after acute viral infection (35), as well the emergence of T cell responses to other (often weaker) epitopes during chronic infection (40) in which higher viral antigenic concentrations are reached within an inflammatory microenvironment in association with prolonged Ag cross-presentation. However, these CTL responses were only detectable following in vitro restimulation with subdominant peptide-pulsed APC in the absence of the dominant peptide. Examination of CTL responses in vivo showed that subdominant-specific effector CTL could not be detected in association with strong dominant-specific functional CTL, implying either that these precursors did not become fully functional in vivo or that CTL recognizing the dominant epitope are potent suppressors, as suggested by others (13). It has been suggested that cryptic epitopes may be revealed when there is a sufficiently strong and prolonged inflammatory or humoral response. However, in these experiments, despite the induction of significant responses to other epitopes and detectable levels of circulating anti-OVA Abs (data not shown), no response to a cryptic epitope was unmasked.
In conclusion, we have shown that the threshold levels of Ag concentration required for the generation of dominant epitope-specific CTL, after cross-presentation, are low, and that the in vivo duration of their lytic activity parallels the kinetics of Ag presentation. In contrast, generating CTL to a weaker epitope requires a high Ag dose and persisting Ag cross-presentation. These findings have important implications for vaccination and immunotherapeutic strategies, and we are currently studying some of these issues using our tumor immunology models (9, 10).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Delia Nelson, Department of Medicine, University of Western Australia, Queen Elizabeth II Medical Center, 4th Floor, G Block, Nedlands, Western Australia, Australia, 6009.
3 Abbreviations used in this paper: DLN, draining lymph node; CFSE, 5,6-carboxy-fluorescein succinimidyl ester; CM, complete medium; DC, dendritic cell.
Received for publication May 25, 2000. Accepted for publication August 29, 2000.
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, 20 µg by •, and 2 µg by 
, respectively) were in vitro
restimulated with APC pulsed with 10-6 M SIINFEKL. Five
days later, a standard 51Cr release in vitro CTL assay was
performed on EL4 target cells pulsed with the same peptide. Controls
included mice given PBS only (
). Data are from one representative
experiment of three, all of which showed the same pattern.
