A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo

Helen Beacock-Sharp1, Anne M. Donachie1, Neil C. Robson1 and Allan M. Mowat1

1 Department of Immunology and Bacteriology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, UK

Correspondence to: A. M. Mowat; E-mail: a.m.mowat{at}clinmed.gla.ac.uk
Transmitting editor: A. Cooke


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Immune-stimulating complexes (ISCOMS) are adjuvant vectors which are unusual in being able to prime both CD4+ and CD8+ T cells by parenteral and mucosal routes. However, their mode of action is unclear and to define better the cellular interactions involved we have studied the ability of ISCOMS containing ovalbumin (OVA) to prime TCR transgenic CD4+ or CD8+ T cells in vivo. Immunization with OVA ISCOMS caused activation and clonal expansion of CD4+ and CD8+ T cells in the T cell areas of the draining lymph nodes, followed by the migration of both CD4+ and CD8+ T cells into the B cell follicle. The T cells were primed to proliferate and secrete IFN-{gamma} after re-stimulation in vitro with the appropriate OVA peptide and CD8+ T cell priming occurred in the absence of CD4+ T cells. Increasing the number of dendritic cells (DC) in vivo with flt3 ligand augmented the expansion and activation of the OVA-specific T cells, particularly CD8+ T cells. These studies indicate DC play a central role in the priming of both CD4+ and CD8+ T cells in vivo, and suggest that an ability to target DC may allow ISCOMS to be powerful vaccine vectors for stimulating protective immunity.

Keywords: adoptive transfer, CD4+ T cell, CD8+ T cell


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A major goal of current research is the development of orally active subunit vaccines which can induce both mucosal and systemic immunity. However, purified protein and peptide antigens are poorly immunogenic unless administered with an adjuvant, especially when administered by mucosal routes (1). A number of candidate mucosal adjuvants have been described, including recombinant organisms such as attenuated Salmonella or the related cholera toxin and the heat-labile enterotoxin of Escherichia coli. However, their use is restricted by potential toxicity, the limited range of effects induced and the fact that such vectors are themselves immunogenic (2).

Immune-stimulating complexes (ISCOMS) are non-antigenic particulate vectors composed of cholesterol, phosphatidylcholine and the saponin adjuvant Quil A (3). Protein antigens incorporated into these rigid cage-like structures are strongly immunogenic by oral and parenteral routes, stimulating a wide range of immune responses in vivo. These include systemic delayed-type hypersensitivity responses, serum IgG1 and IgG2a and intestinal secretory IgA antibodies, and the production of Th1- and Th2-dependent cytokines (4,5). In addition, ISCOMS have a potent ability to prime MHC class I-restricted cytotoxic T lymphocyte (CTL) responses in vivo. Together, these attributes support the view that ISCOMS may be useful oral vaccine vectors for inducing protective immunity against a number of different pathogens. However, for this goal to be realized, it will be important to understand better how and where ISCOMS prime antigen-specific T cells, as well as to investigate the nature of the antigen-presenting cell (APC) involved. We have found recently that ISCOMS recruit and activate a number of components of the innate immune response, including macrophages and dendritic cells (DC). This is followed by the accumulation of CD4+ and CD8+ T cells, some of which are activated as shown by expression of CD25 (6). However, it was not determined if these were antigen-specific T cells nor how the initiation of specific CD4+ and CD8+ T cell responses to ISCOMS related to the recruitment of the innate immune system, and of APC in particular. Here we have explored the cellular and anatomical basis of antigen-specific T cell priming by ISCOMS by monitoring the activation of adoptively transferred OVA-specific TCR transgenic CD4+ and CD8+ T cells after s.c. immunization with OVA in ISCOMS. In addition, as DC are known to be the most efficient APC for presenting antigens to naive T cells and are recruited to the site of injection of ISCOMS (6), we have investigated the role of DC in the priming of T cells by antigens incorporated into ISCOMS. To do this, we made use of the ability of the cytokine flt3 ligand (flt3L) to produce a selective increase in the number of DC in vivo (7). Our results show that immunization with OVA ISCOMS results in the activation and expansion of antigen-specific CD4+ and CD8+ effector T cells in the draining lymph node (LN), and these effects were enhanced by treating mice with flt3L. These studies highlight the potential usefulness of ISCOMS as practical vaccine vectors.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Specific pathogen-free female BALB/c (H-2d) and C57Bl/6 (H-2b) mice were obtained from Harlan Olac (Bicester, UK). DO11.10 mice, transgenic for a TCR specific for the I-Ad-presented OVA peptide 323–339 (8), were obtained originally from Dr N. Lycke (Department of Clinical Immunology, University of Göteborg, Sweden). OT-1 mice transgenic for a TCR specific for the H-2Kb-presented OVA peptide 257–264 (9) were obtained from Dr M. Merkenschlager (Clinical Sciences Centre, Hammersmith Hospital, London, UK). All these mice were bred at the University of Glasgow under specific pathogen-free conditions. MHC class II–/– mice (H-2b) (10), obtained from Dr M. Merkenschlager, were bred and maintained under isolator conditions. All mice were used from 8 weeks of age.

Treatment with flt3L
Mice were injected i.p. daily with 10 µg of flt3L (a kind gift of Immunex, Seattle, WA) in 0.2 ml saline for 9 days before immunization. Control mice were injected with saline alone.

Preparation of OVA ISCOMS
ISCOMS containing OVA were prepared using cholesterol, phosphatidylcholine and Quil A, as described previously (11).

In vivo priming of adoptively transferred OVA-specific TCR transgenic T cells
The method first described by Kearney et al. was adapted (12). Spleen, and popliteal, inguinal, brachial, cervical, para-aortic and mesenteric LN were harvested from DO11.10 or OT-1 mice, and single-cell suspensions prepared in sterile RPMI 1640 medium (Gibco/BRL, Paisley, UK) by homogenization through Nitex mesh (Cadisch, London, UK). After centrifugation (400 g, 5 min), the cells were resuspended and counted using phase-contrast microscopy to exclude dead cells. The cell preparations were stained with anti-CD4 (PharMingen, San Diego, CA) and KJ1.26 (purified hybridoma supernatant) mAb or with anti-CD8 and anti-V{alpha}2 mAb (both PharMingen) to determine the proportion of T cells expressing the DO11.10 or OT-1 transgenic TCR respectively. TCR transgenic DO11.10 or OT-1 T cells (3 x 106) were injected i.v. into syngeneic BALB/c or B6 mice respectively and the recipients were immunized 2–3 days later with 5 µg OVA in ISCOMS by s.c. injection into a footpad.

Depletion of CD4+ cells in vitro
For transfer into MHC class II–/– mice, lymphocytes from OT-1 mice were depleted of CD4+ T cells by incubating 1 x 107 lymphoid cells/ml with 200 µg/ml purified GK1.5 anti-CD4 in culture medium (RPMI 1640 containing 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin; all Gibco/BRL) for 30 min at 4°C. After washing, 10% rabbit complement (Low-Tox-M; VH-Bio, Newcastle, UK) in culture medium was added and the cells incubated for a further 30 min at 37°C. The depletion process was repeated and the purity of the cells assessed by flow cytometry. The population comprised 65% CD8+V{alpha}2+ cells with <1.5% contaminating CD4+ cells.

Two- and three-colour flow cytometry
Aliquots of 1–10 x 105 cells were washed in FACS buffer (PBS containing 2% FCS and 0.05% sodium azide) and incubated for 30 min at 4°C in 100 µl anti-CD16/CD32 antibody (Fc Block; PharMingen) to block non-specific binding. After washing in FACS buffer, combinations of biotinylated and fluorochrome-labelled mAb were added in 100 µl of FACS buffer, and incubated for 30 min at 4°C in the dark. After washing twice, the cells were incubated for 30 min at 4°C in 100 µl of streptavidin conjugated to phycoerythrin (PE) or PerCP (both PharMingen) to visualize the biotinylated antibodies. The mAb used were PerCP–anti-CD4, PE–anti-CD8, biotinylated anti-V{alpha}2, FITC–anti-CD69 and FITC–anti-CD25 (all PharMingen). Bio-KJ1.26 antibody was prepared from hybridoma supernatant. The cells were analysed on a Becton Dickinson (Oxford, UK) FACScan flow cytometer using CellQuest software.

Measurement of OVA-specific proliferation in vitro
The popliteal LN draining the site of immunization was harvested at various time points after immunization and a single-cell suspension prepared. Aliquots of 2 x 105 cells in 200 µl culture medium were cultured in triplicate in flat-bottomed microtitre plates (Costar, High Wycombe, UK) in the presence or absence of 10 µg/ml OVA peptides 323–339 or 257–264 (Sigma Genosys, Cambridge, UK). After incubation for 48 h at 37°C in 5% CO2, the cells were pulsed with 1 µCi [3H]thymidine (West of Scotland Radionucleotide Dispensary, Glasgow, UK) and incubated for a further 18 h prior to harvesting.

Measurement of OVA-specific cytokine production in vitro
Supernatants were harvested after 48 h of lymphocyte culture, centrifuged at 13,000 g and stored at –20°C until required. IFN-{gamma} and IL-5 levels were determined by sandwich ELISA, as described previously (13).

Measurement of OVA-specific CTL activity
OVA-specific CTL activity in ISCOMS-primed mice transferred with OT-1 T cells was measured directly ex vivo by culturing draining LN cells in 200 µl culture medium with 5 x 103 51Cr-labelled EL4 cells pulsed with 10 µg/ml OVA 257–264 peptide at E:T ratios of 100:1, 50:1 and 25:1. After 4 h incubation, 100 µl supernatant was removed and 51Cr-specific radioactivity measured in a {gamma}-counter (Compugamma; Wallac, Turku, Finland). Specific CTL activity was calculated using the following formula: specific lysis = [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x 100%.

Triton X (10%) (Sigma, Poole, UK) was used to obtain maximum release, while spontaneous release was obtained from target cells cultured in medium alone. Control wells contained EL4 cells without OVA peptide.

To examine for the presence of primed CTL precursors in vivo, LN cells isolated from ISCOMS-immunized mice were re-stimulated with EG7.OVA cells for 5 days in vitro prior to measurement of CTL activity, as described previously (11).

Visualization of OVA-specific T cells and DC in tissues by immunohistochemistry
Immunohistochemical analysis of the draining LN was performed as described previously (14). Briefly, LN were frozen in OCT embedding medium (Bayer, Newbury, UK), and 6-µm sections were cut and air-dried before fixation in acetone. Sections were blocked using an avidin/biotin blocking kit (Vector, Peterborough, UK) prior to staining. To detect DO11.10 and OT-1 cells, sections were incubated with 100 µl biotinylated KJ1.26 or anti-V{alpha}2 respectively, diluted 1/500 in PBS/2% normal goat serum. Negative control sections were incubated with PBS/2% normal goat serum alone. The primary antibodies were detected using avidin–biotin complex conjugated to alkaline phosphatase (ABC-AP; Vector) followed by 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT; Vector), resulting in blue staining of transgenic T cells. To stain for a second cell population, tissues were incubated with 100 µl biotinylated antibody reactive with either B cells (B220; PharMingen) or DC (anti-CD11c; PharMingen) at the appropriate concentration diluted in PBS/2% normal goat serum. These antibodies were detected using 100 µl avidin–biotin complex conjugated to horseradish peroxidase (ABC–HRP; Vector) which was visualized using 3,3'-diaminobenzidine (Vector).

Statistics
Where appropriate, results are expressed as mean ± SD and compared using Student’s t-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effects of expanding DC numbers on the in vivo clonal expansion and activation of OVA-specific CD4+ T cells after priming with OVA ISCOMS
In the first experiments, OVA-specific CD4+ T cells from DO11.10 mice were transferred into BALB/c mice and the expansion of TCR transgenic T cells followed in the draining popliteal LN at intervals after immunization with 5 µg OVA in ISCOMS. The proportion of TCR transgenic T cells, as assessed by flow cytometric detection of CD4+KJ1.26+ cells, remained <1% of the LN population in both unimmunized and flt3L-treated unimmunized animals (data not shown). In two separate experiments, immunization of normal mice with OVA ISCOMS led to an increase in the proportion of DO11.10 T cells which peaked on day 4, when the mean proportion of transgenic CD4+ T cells was ~3-fold above that in unimmunized mice. These levels remained higher than in unimmunized controls until the end of the study on day 7 (Fig. 1A). As illustrated by the experiment shown in Fig. 1(B), there was a parallel increase in the absolute number of DO11.10 T cells in the draining LN of ISCOMS-immunized mice which also peaked on day 4.



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Fig. 1. Adoptively transferred DO11.10 cells are activated and expand after immunization with OVA ISCOMS. BALB/c mice were adoptively transferred with 3 x 106 CD4+KJ1.26+ cells from DO11.10 mice 2 days prior to immunization with 5 µg OVA in ISCOMS (solid symbols) or no immunization (open symbols). Mice were treated with flt3L (squares) or saline (circles) for 9 days before immunization. (A) The proportion of CD4+KJ1.26+ cells was determined in the draining popliteal LN by flow cytometry and the fold increase compared with that seen in adoptively transferred, unimmunized controls was calculated. The mean ± SD of four animals from two pooled experiments is shown (*P = 0.005 versus control immunized mice). (B) The absolute number of the TCR transgenic T cells, (C) the size, and (D) expression of CD69 and (E) CD25 on OVA-specific CD4+ T cells was determined. Panels (B)–(E) show the mean ± range of two individual mice from one of the two replicate experiments summarized in (A). Similar results were found in each case.

 
To assess the activation status of the T cells that had responded to OVA ISCOMS, their size and expression of CD69 and CD25 was analysed in both experiments, one of which is shown in Fig. 1(C–E). The size of the CD4+KJ1.26+ cells from immunized mice, as assessed by forward scatter, was already greatly increased on day 1 compared with those from unimmunized mice (Fig. 1C). Cell size declined on subsequent days, but the very rapid activation of DO11.10 T cells was confirmed by the expression of CD69 on >40% of CD4+KJ1.26+ cells taken 1 day after immunization with OVA ISCOMS (Fig. 1D). More than 20% of the OVA-specific CD4+ T cells also expressed CD25 within 1 day of immunization and this was maintained until day 2, before declining gradually thereafter (Fig. 1E).

To assess the effects of increasing the number of DC on the expansion and activation of CD4+ T cells, mice were treated with flt3L for 9 days before immunization. In the two experiments summarized in Fig. 1(A), this resulted in significantly enhanced expansion of DO11.10 cells compared with that found in immunized control mice, with the proportion of CD4+KJ1.26+ T cells rising to levels 2–3 times those in immunized control recipients at the peak of expansion on day 4 (P = 0.005). The time course of expansion was not altered by treatment with flt3L. A similar effect of flt3L was also evident when the absolute number of DO11.10 T cells was analysed in the experiment shown in Fig 1(B–E). In addition, the proportion of DO11.10 T cells expressing CD69 was now 55% (Fig. 1D). CD25 was also expressed on >40% of the CD4+KJ1.26+ cells in flt3L-treated mice compared with 25% in control recipients (Fig. 1E). There were no differences in the size of the OVA-specific CD4+ T cells in flt3L-treated and control mice (Fig. 1C). The effects of flt3L were identical in the repeat experiment included in Fig. 1(A) and we concluded that increasing the number of DC enhances the expansion of CD4+ T cells by ISCOMS-associated antigen.

Effects of expanding DC numbers on the in vivo clonal expansion and activation of OVA-specific CD8+ T cells after priming with OVA ISCOMS
Very little is known about the unusual ability of ISCOMS to enable exogenous antigens to prime CD8+ T cells, and in our second series of experiments we used the adoptive transfer model and flt3L to study CD8+ T cell priming in more detail.

The expansion and activation of CD8+ TCR transgenic T cells was tracked by monitoring the numbers of CD8+V{alpha}2+ cells in the draining LN. In the two separate experiments summarized in Fig. 2(A), immunization of normal recipients with OVA ISCOMS led to an increase in the percentage of OT-1 T cells which peaked at ~5-fold that seen in unimmunized mice on day 4 and returned to basal levels by day 7 (Fig. 2A). In the individual experiment illustrated in Fig. 2(B–E), the absolute numbers of CD8+V{alpha}2+ T cells also increased between days 2 and 4, and then declined to the starting level by day 7 (Fig. 2B). Increasing the number of DC with flt3L resulted in an increase in the percentage of OT-1 cells over that seen in flt3L-treated unimmunized mice, which at its peak averaged >20-fold in the two experiments summarized in Fig. 2(A) and was significantly greater than the expansion seen in control immunized mice at this time (P = 0.04). Indeed, in some flt3L-treated animals, 60% of the total mononuclear cell population of the popliteal LN comprised TCR transgenic CD8+ T cells at the peak of the response on day 4. This effect of flt3L on CD8+ T cell expansion was also reflected in the absolute numbers of CD8+V{alpha}2+ T cells (Fig. 2B).



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Fig. 2. Adoptively transferred OT-1 cells are activated and expand after immunization with OVA ISCOMS. B6 mice were adoptively transferred with 3 x 106 CD8+V{alpha}2+ cells from OT-1 mice 2 days prior to immunization with 5 µg OVA in ISCOMS (solid symbols) or no immunization (open symbols). Mice were treated with flt3L (squares) or saline (circles) for 9 days before immunization. (A) The proportion of CD8+V{alpha}2+ cells was determined in the draining popliteal LN by flow cytometry and the fold increase compared with that seen in adoptively transferred, unimmunized controls was calculated. The mean ± SD of four animals from two pooled experiments is shown (*P = 0.04 versus control immunized mice). (B) The absolute number of the TCR transgenic T cells, (C) the size, and (D) expression of CD69 and (E) CD25 on OVA-specific CD8+ T cells was determined. Panels (B)–(E) show the mean ± range of two individual mice from one of the two replicate experiments summarized in (A). Similar results were found in each case.

 
The clonal expansion of OT-1 T cells in immunized control mice was accompanied by increased cell size, which was maximal 3 days after immunization (Fig. 2C) and was preceded by rapid up-regulation of CD69 expression (Fig. 2D). This peaked 24 h after immunization and decreased to control levels by day 3, before increasing again on day 7. Expression of CD25 by OVA-specific CD8+ T cells was evident 2 days after immunization with peak expression on day 3 and nearly all CD25 expression was lost by day 7. Interestingly, the increased expansion of OVA-specific CD8+ T cells in flt3L-treated immunized recipients was accompanied by a substantial increase in the size of these cells at the peak of the response compared with those in control-immunized recipients (median of 800 versus 600; Fig. 2C). At this time point, >60% of the OVA-specific CD8+ T cells also expressed CD25 in flt3L-treated, ISCOMS-immunized mice, compared with only 20% in control-immunized mice (Fig. 2E). The high percentage of OVA-specific CD8+ T cells expressing CD69 in immunized controls was not affected substantially by flt3L treatment, but this persisted for up to 24 h longer in flt3L-treated recipients (Fig. 2D). Thus, an increase in the number of DC had a dramatic effect on the priming of specific CD8+ T cells by ISCOMS.

Effects of expanding DC numbers on the functional responses of OVA-specific CD4+ and CD8+ T cells primed with OVA ISCOMS
To assess further the effects of DC on the priming of CD4+ and CD8+ T cells by OVA ISCOMS, we re-stimulated draining LN cells with appropriate OVA peptides in vitro, and measured proliferation and IFN-{gamma} and IL-5 secretion. CTL activity was also determined in recipients of OT-1 cells.

Immunization of BALB/c mice given DO11.10 T cells with OVA ISCOMS induced high proliferative recall responses to OVA 323–339 which were greatest on day 2 after immunization and declined to background by day 7 (Fig. 3A). These T cells were also primed for IFN-{gamma} secretion (Fig. 3B), which was greatest on day 2 after immunization and was still greater than that in unimmunized control mice on day 4. Flt3L treatment slightly enhanced the proliferative response to OVA 323–339 in OVA ISCOMS-immunized mice at all times, but did not alter the kinetics of the response (Fig. 3A). Treatment with flt3L did not increase the amount of IFN-{gamma} detected at the peak of the response, but slightly more IFN-{gamma} was detectable at the later time points (Fig. 3B). There was no IL-5 production in any of the groups (data not shown). Similar results were obtained in a repeat experiment.



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Fig. 3. Antigen-specific proliferation and IFN-{gamma} secretion after immunization with OVA ISCOMS. BALB/c mice were adoptively transferred with 3 x 106 CD4+KJ1.26+ cells from DO11.10 mice 2 days prior to immunization with 5 µg OVA in ISCOMS (solid symbols) or no immunization (open symbols). Mice were treated with flt3L (squares) or saline (circles) for 9 days before immunization. At the indicated time after immunization the popliteal LN was taken, and a single-cell suspension prepared and re-stimulated in vitro with OVA 323–339. Antigen-specific proliferation (A) and IFN-{gamma} secretion (B) was measured, and the results shown are the mean of triplicate cultures ± range from two mice per group. The data are representative of two replicate experiments.

 
Immunization with OVA ISCOMS also primed OT-1 T cells transferred into B6 mice to proliferate and produce IFN-{gamma} when stimulated in vitro with OVA 257–264. These responses were first detectable on day 3 after immunization, peaked on day 4 and had returned to background levels by day 7 (Fig. 4A and B). Both these responses were enhanced in flt3L-treated recipients. IL-5 could not be detected in the supernatants of any of these cultures (data not shown).



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Fig. 4. Antigen-specific proliferation and IFN-{gamma} secretion after immunization with OVA ISCOMS. B6 mice were adoptively transferred with 3 x 106 CD8+V{alpha}2+ cells from OT-1 mice 2 days prior to immunization with 5 µg OVA in ISCOMS (solid symbols) or no immunization (open symbols). Mice were treated with flt3L (squares) or saline (circles) for 9 days before immunization. At the indicated time after immunization the popliteal LN was taken, and a single-cell suspension prepared and re-stimulated in vitro with OVA 257–264. Antigen-specific proliferation (A) and IFN-{gamma} secretion (B) was measured, and the results shown are the mean of triplicate cultures ± range from two pooled mice per group. CTL activity to 51Cr-labelled EL4 cells pulsed with OVA 257–264 was determined on day 1 after immunization ex vivo (C), or after stimulation with EG7.OVA cells in vitro (D) or ex vivo on day 5 (E). CTL activity to EL4 cells not pulsed with OVA 257–264 was <5% in all instances. Results shown are the mean of triplicate cultures of two pooled mice per group and are representative of two replicate experiments.

 
We also examined the ability of the LN cells from OT-1 transferred mice to lyse EL4 cells pulsed with OVA 257–264, both before and after re-stimulation with OVA-expressing EG7.OVA cells in vitro. Freshly isolated cells from immunized mice showed low levels of specific CTL activity against peptide-pulsed EL4 cells at all E:T ratios when tested on day 1. This CTL activity was approximately doubled in flt3L-treated recipients, even though the number of CD8+V{alpha}2+ cells was approximately equal in the two groups at this time (Fig. 4C). Specific CTL activity was also apparent when LN cells from mice immunized 1 day before were re-stimulated in vitro with EG7.OVA cells and this was enhanced by up to 50% in flt3L-treated mice (Fig. 4D). Five days after immunization with OVA ISCOMS, some lysis of OVA 257–264-pulsed target cells by freshly isolated LN cells could still be detected.

Priming of OVA-specific CD8+ T cells by OVA ISCOMS does not require CD4+ T cell help
In view of the potent priming of CD8+ T cells by OVA ISCOMS, we thought it important to investigate the requirement for CD4+ T cell help in this process. Thus, CD4+ T cell-depleted OT-1 cells were transferred into syngeneic MHC class II-deficient animals which were then immunized with OVA ISCOMS as before.

Immunization with OVA ISCOMS resulted in marked expansion of OT-1 cells in MHC class II knockout mice, with similar kinetics and magnitude to that found in intact recipients (Fig. 5A and B). Indeed, at the peak of the response on day 4, ~50% of the LN cells were CD8+V{alpha}2+ cells. The expansion of OT-1 cells in class II MHC knockout recipients was accompanied by the expression of CD69 (Fig. 5C), blast transformation and CD25 expression (data not shown) with kinetics analogous to wild-type recipients.



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Fig. 5. CD8+ T cell priming by OVA ISCOMS occurs in the absence of CD4+ T cell-mediated help. OT-1 cells were depleted of CD4+ cells and transferred into MHC class II-deficient H-2b animals, which were then immunized in a footpad with 5 µg OVA in ISCOMS (solid circles) or left unimmunized (open circles). The proportion of TCR transgenic lymphocytes (A), absolute number (B) and percentage of CD69-expressing CD8+V{alpha}2+ cells (C) was determined by flow cytometry. The mean results ± range of two individual mice are shown. Proliferative responses to OVA 257–264 were determined 3 days after immunization (D) of wild-type B6 and MHC class II-deficient animals transferred with CD4-depleted OT-1 cells. Results shown are the mean ± SD of triplicate cultures from two pooled mice per group. IFN-{gamma} secreted by popliteal LN cells stimulated in vitro was also measured (E). Results shown are the mean of triplicate cultures ± range from two pooled mice per group and are representative of two replicate experiments.

 
The ability of ISCOMS to prime CD8+ T cells in MHC class II knockout animals was confirmed by the fact that LN cells from these mice showed excellent proliferative responses and produced IFN-{gamma} in response to in vitro stimulation with OVA 257–264 (Fig. 5D and E). Thus priming of antigen-specific CD8+ T cells by ISCOMS does not require MHC class II-restricted CD4+ T cell help.

Localization of OVA-specific T cell expansion in vivo
Previous studies using adoptive transfer of TCR transgenic CD4+ T cells have shown that one of the most important effects of adjuvants which promote T cell expansion and activation in vivo is to stimulate migration of these T cells into B cell follicles (12,15). Therefore, to investigate further the anatomical events involved in the activation of antigen-specific T cells by ISCOMS, we examined whether a similar process occurred after immunization with OVA ISCOMS.

Only a small number of KJ1.26+ cells could be seen in the T cell-dependent area (TDA) of the LN of unimmunized mice that had been transferred with DO11.10 cells (Fig. 6A and D). After immunization with OVA ISCOMS, expansion of the OVA-specific CD4+ T cells (Fig. 6B and F) was seen first in the TDA on days 1–2, where CD11c+ DC were also present (Fig. 6F). By days 2–3, OVA-specific CD4+ T cells were still present in the TDA, but could also now be seen migrating into the B cell-rich follicles (Fig. 6B). Interestingly, the expansion of OVA-specific CD8+ T cells followed a similar anatomical pattern, in which V{alpha}2+ T cells first appeared in markedly increased numbers adjacent to CD11c+ DC in the TDA on days 2–3 (data not shown) and subsequently some of these cells migrated into the B cell-rich follicles (Fig. 6I and J).



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Fig. 6. Anatomical visualization of ISCOMS-primed, OVA-specific CD4+ and CD8+ T cells in vivo. Mice were treated with flt3L or saline and adoptively transferred with 3 x 106 CD4+KJ1.26+ (A–G) or CD8+V{alpha}2+ (H–J) cells, 2 days before immunization with 5 µg of OVA in ISCOMS (B, C, F, G, I and J), or were left unimmunized (A, D, E and H). Sections (6 µm) of the draining popliteal LN taken 2 (A–G) or 3 (H–J) days after immunization were stained with KJ1.26 (blue in A–G) or V{alpha}2 antibodies (blue in H–J) to detect OVA-specific CD4+ and CD8+ T cells, respectively. B cells were detected using B220 (brown in A–C and H–J) and DC by staining for CD11c (brown in D–G). Panel (J) is a higher magnification of (I), showing an area within the B cell follicle.

 
The immunohistochemical analysis confirmed the great expansion of CD11c+ DC in the popliteal LN of flt3L-treated mice (Fig. 6E and G), but the disruption of normal lymphoid architecture caused by this expansion of DC prevented assessment of how the increase in DC altered the anatomy of CD4+ or CD8+ T cell expansion (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The experiments presented here are the first to study the ability of a clinically proven vaccine vector to prime antigen-specific T cells at the cellular level. We have demonstrated that immunization with OVA ISCOMS simultaneously primes adoptively transferred OVA-specific TCR transgenic CD4+ and CD8+ T cells for a wide range of antigen-specific effector functions. This resulted from rapid clonal expansion of CD4+ and CD8+ T cells in the TDA of the draining LN followed by their migration into the B cell follicles. Expansion and activation of the OVA-specific CD8+ T cells occurred normally in the absence of MHC class II-restricted CD4+ T cells. Increasing the number of DC by treatment with flt3L resulted in an enhancement of both CD4+ and CD8+ T cell responses, implying an important role for DC in the presentation of ISCOMS-associated antigen.

Adoptive transfer of phenotypically identifiable TCR transgenic T cells into normal mice allows the priming of these T cells to be tracked in vivo without resorting to the unphysiological level which occurs in an intact TCR transgenic mouse (16). To date this technique has been used primarily to investigate the responses of CD4+ or CD8+ T cells in isolation, after immunization with experimental adjuvants or recombinant virus vectors (15,17,18). The ability of OVA ISCOMS to induce rapid activation and clonal expansion of DO11.10 T cells, followed by their migration into the B cell follicle, is analogous to that reported using complete Freund’s adjuvant (CFA), lipopolysaccharide or DNA microparticles as adjuvants in the adoptive transfer model (15,1820). ISCOMS may be even more efficient than these adjuvants, as extremely low amounts of antigen were administered in ISCOMS compared with that used in the other systems [5 µg in ISCOMS compared to 100–2000 µg with CFA or lipopolysaccharide (15,18,20)].

ISCOMS were also very efficient at priming adoptively transferred CD8+ T cells in vivo. The behaviour of adoptively transferred CD8+ T cells has not previously been examined after administration of native protein in adjuvant, but our results are consistent with studies using peptide in CFA (21), endogenous self-peptides (22), native proteins alone (23) or virally encoded antigens (17,24). Indeed, the kinetics of activation following immunization with OVA ISCOMS were very similar to that reported using OVA-expressing VSV as a vector, although unsurprisingly, the magnitude of the response was somewhat greater using the replicating virus (17). The clonal expansion of OT-1 cells in vivo and the antigen-specific effector functions we detected are consistent with the ability of ISCOMS to prime MHC class I-restricted CTL in normal mice (11,2527). However, we were previously unable to detect antigen-specific proliferation of CD8+ T cells in ISCOMS-immunized wild-type mice (27), highlighting the sensitivity of the adoptive transfer model for the direct study of antigen-specific T cells.

ISCOMS-primed CD8+ T cell responses were normal when OT-1 cells were transferred into MHC class II-deficient hosts, indicating that CD4+ T cell-mediated help was not required for these responses. CD4+ T cells are required for effective priming of CD8+ T cells in other circumstances, either as a source of IL-2 (28,29) or to activate APC to express sufficient co-stimulatory molecules (30,31). Our results suggest that ISCOMS share the properties of some viruses and adjuvants such as CFA (30) or the heat-labile enterotoxin of E. coli (32) which can overcome the requirement for CD4+ T cells, presumably by directly activating the APC to express sufficient co-stimulatory molecules. We are currently investigating the ability of ISCOMS to up-regulate expression of co-stimulatory molecules and inflammatory cytokines by the APC. An alternative explanation could be that the transfer of a relatively high number of TCR transgenic T cells may abrogate the need for CD4+ T cell help in vivo (33) and this needs to be investigated using graded doses of transferred T cells.

The efficiency of ISCOMS in priming antigen-specific CD4+ T cells was underlined by the fact that their clonal expansion was followed by migration into the B cell follicles, a phenomenon which occurs only when immunogenic adjuvants have been used (12,15). A novel feature of our study was that we observed similar migration of OVA-specific CD8+ T cells into the B cell follicles. CD8+ T cells have been identified in splenic germinal centres of HIV patients (34), and CD8+ T cell clones which have the ability of provide help for the production of IgM and IgG have been isolated from allogeneic-stimulated murine splenocytes (35). Thus, the OT-1 cells within the B cell follicles may be assisting in the production of OVA-specific antibodies. An alternative possibility is that the CD8+ T cells may kill B cells expressing antigen on their cell surface.

The ability of flt3L to enhance the priming of both CD4+ and CD8+ T cells is consistent with a central role for DC in the presentation of ISCOMS-associated antigen. Treatment with flt3L is known to increase the numbers of several subtypes of CD11c+ DC, including CD11bbright, CD11bdull and CD11b, and both CD8{alpha}+ and CD8{alpha} DC (36). It was beyond the scope of this study to define the subtype(s) of DC responsible for presenting ISCOMS-associated antigen to T cells in vivo, but preliminary studies indicate that CD8{alpha} DC derived from bone marrow can present OVA ISCOMS to both DO11.10 and OT-1 T cells in vitro (N. Robson, in preparation). We are currently studying the role of different DC subsets in the presentation of ISCOMS-associated antigen in vivo. Previous studies have shown that flt3L treatment can enhance CD4+- and CD8+-dependent immune responses in vivo (3741), although ours is the first study to examine antigen-specific T cell responses directly. As flt3L is not itself a DC-activating adjuvant (37), the enhanced priming of CD4+ and CD8+ T cells by ISCOMS in flt3L-treated mice suggests that ISCOMS may activate DC in vivo. However, the mechanism by which exogenous ISCOMS-associated antigen is presented to CD8+ T cells remains to be elucidated. It may be that ISCOMS gain access to the cytoplasmic-processing pathway either by fusing directly with the cell membrane or with intracellular membranes after uptake into endocytic compartments. Alternatively, as ISCOMS are known to be toxic to some cells and DC have been shown to be effective at cross-presentation of antigen from cells which were loaded with antigen and then died (42), it may be that cross-presentation of ISCOMS-associated antigen could be occurring. However, our current experiments show that bone marrow-derived DC can present ISCOMS-associated antigen directly to CD8+ T cells in vitro, supporting the idea that cross-presentation may not be required in vivo (N. Robson, in preparation). It is unclear why flt3L treatment had a less dramatic effect on the CD4+ T cell response, although it could reflect the fact that the presentation of ISCOMS to CD4+ T cells by DC is already approaching optimal levels and cannot be influenced by flt3L treatment. Alternatively, ISCOMS may not be able to activate the increased number of DC to the extent needed to efficiently prime CD4+ T cells. It is also possible that cells other than DC could be involved in presentation of ISCOMS to CD4+ T cells and we are currently investigating these ideas in more detail.

In conclusion, the experiments presented here confirm at the cellular level the ability of ISCOMS to prime CD4+ and CD8+ T cell responses, underlining their potential as adjuvant vectors for novel subunit vaccines against a wide range of intracellular and extracellular pathogens. These effects may be particularly dependent on DC, suggesting that targetting ISCOMS for presentation by DC may further augment the resulting immune responses.


    Acknowledgements
 
This work was supported by The Wellcome Trust, and by grants BIO4-CT98-0505 and QLK2-CT-1999-0228 from the EC Biotechnology Programme Frameworks 4 and 5. We thank Dr Paul Garside for critical reading of the manuscript.


    Abbreviations
 
APC—antigen-presenting cell

CFA—complete Freund’s adjuvant

CTL—cytotoxic T lymphocyte

DC—dendritic cell

flt3L—flt3 ligand

ISCOMS—immune-stimulating complexes

LN—lymph node

PE—phycoerythrin

OVA—ovalbumin

TDA—T cell-dependent area


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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