Soluble proteins and haptens on bone marrow-derived dendritic cells are presented to host CD4 T cells in an MHC-restricted manner
Edit B. Olasz1,
Jay Linton1 and
Stephen I. Katz1
1 Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1908, USA
Correspondence to: S. I. Katz; E-mail: skatz{at}box-s.nih.gov
Transmitting editor: A. Falus
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Abstract
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Because of their potent antigen-presenting capacity, dendritic cells (DC) have been used extensively in immunotherapy protocols. Our purpose was to functionally characterize mouse bone marrow-derived DC (BMDC) in vitro (in protein antigen- and hapten-specific assays) and in vivo (injecting soluble protein- and hapten-pulsed DC) to determine their suitability for the generation of Th cell responses. Furthermore, we determined whether there is cross-presentation on MHC class II molecules during in vivo protein and hapten sensitization. Co-culture of protein-pulsed [with hen egg lysozyme (HEL) or with pigeon cytochrome c (CYT)] DC with T cells from HEL- or CYT- sensitized mice induced antigen-specific T cell proliferation, but compared to cultured Langerhans cells (LC), BMDC required higher protein antigen-pulsing concentrations (100 µg and 1 mg/ml). In contrast, at low protein concentrations (10 µg/ml), BMDC stimulated an HEL-specific hybridoma very efficiently. Using an in vitro T cell proliferation assay and in vivo delayed-type hypersensitivity and contact sensitivity assays, we found that protein- and hapten-pulsed BMDC were able to sensitize syngeneic but not allogeneic hosts. Furthermore, if we injected BALB/c- and C57BL/6-derived HEL-pulsed BMDC into F1 mice, specific secondary proliferation of primed T cells occurred only when antigen-pulsed stimulator cells syngeneic to the injected BMDC were used. Using this model system we found that soluble proteins and haptens are presented by injected BMDC to host T cells in an MHC-restricted manner in vivo.
Keywords: antigen presentation, contact sensitivity, cross-presentation, delayed-type hypersensitivity, dendritic cell, MHC
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Introduction
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Dendritic cells (DC) are highly efficient antigen-presenting cells (APC) that capture, process and present antigens to naive and primed T cells (1,2). Their ability to initiate potent T cell responses in vivo without any adjuvant has encouraged their utilization for immunization purposes.
Recent studies have focused on the induction of cytotoxic T lymphocyte (CTL) responses with tumor or virus killing activity. In these studies the authors used (37) tumor extracts or tumor RNA (8) which induced antigen-specific CTL mediating tumor immunity. Human trials using DC loaded with tumor antigens have shown (911) promising results. However, using MHC class I-restricted pathways ignores the role of MHC class II-restricted Th cells in initiating and sustaining immune responses.
Thus, our first goal was to functionally characterize bone marrow-derived cells (BMDC) to initiate CD4+ Th cell responses. We used the soluble proteins hen egg lysozyme (HEL) and pigeon cytochrome c (CYT) to study the characteristics of MHC class II-restricted presentation and immunization. The fate of injected DC is not fully known; furthermore, there is very limited knowledge about the phenomenon of cross-presentation. It is known that during both the maintenance of tolerance and the rejection of malignant cells and transplants there is a representation of antigens in association with MHC class I and II products. The mechanism of cross-presentation of peptides presented on MHC class I molecules has recently been shown (12,13) to be transfer of peptide from apoptotic cells to DC. Sauters et al. found that DC phagocytose apoptotic and necrotic cell lines, but only the latter cause potent maturation of DC (14). In contrast of our knowledge about the cross-presentation of antigens on MHC class I molecules, much less is known about presentation of antigens on MHC class II molecules. Recently, Inaba et al., using a mAb that specifically recognizes complexes formed between an MHC class II molecule and a peptide, were able to detect the formation of MHC class IIpeptide complexes in vitro and in vivo after phagocytosis of cell fragments (15). Using in vitro and in vivo functional assays we herein show that BMDC pulsed with intact soluble proteins or haptens induce CD4+ Th responses in an MHC-restricted manner.
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Methods
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Mice
Female BALB/c (I-Ad), C57BL/6 (I-Ab) and F1 (CB6/F1) mice aged 612 weeks old (Animal Production Area, National Institutes of Health, Frederick, MD) were used in all experiments, and were housed under pathogen-free conditions.
Cells
BMDC were generated using the method of Fields et al. (10) with minor modifications. Briefly, BM was flushed from tibias and femurs of female BALB/c or C57BL/6 mice and depleted of red blood cells with ammonium chloride. Cells were plated at 1 x 106 cell/ml in T162 flasks in 50 ml DC medium [DCM: RPMI 1640 containing 5% FCS (Biofluids, Rockville, MD), 50 µM 2-mercaptoethanol (Sigma, St Louis, MO), 10 mM HEPES, pH 7.4, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 20 µg/ml gentamycin (all from Life Technologies, Chagrin Falls, OH), supplemented with 10 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF) and 10 ng/ml IL-4 (PeproTech, Rocky Hill, NJ)]. On day 5, cultures were fed with an additional 25 ml of DCM. On day 7, non-adherent and loosely adherent cells were harvested by vigorous pipetting of cultures. Harvested cells were resuspended in RPMI + 5% FCS and 10 mM HEPES at 5 x 106 cells/ml. Then 10 ml aliquots of this cell suspension were layered onto 15 ml of 14.5% metrizamide (Sigma)/RPMI + 5% FCS and 10 mM HEPES solution in a 50 ml centrifuge tube and centrifuged at 1700 g for 10 min at 4°C. The resulting cell interphase was harvested, washed twice in RPMI + 5% FCS and 10 mM HEPES, and subcultured in DCM for an additional 1 day. On day 8, non-adherent cells were harvested and these cells are referred to as BMDC. The purity of BMDC as determined by FACS analysis was routinely 8595%. Contaminating cells were mostly granulocytes and macrophages. No T or B cells were detected.
Epidermal cells (EC)
Single EC suspensions were obtained from ears of untreated BALB/c or C57BL/6 mice as previously described (16). Fresh LC were obtained by Lympholyte M (Cedarlane, Hornby, Ontario, Canada) gradient centrifugation as previously described (17) or further cultured (1.5 x 106 cells/ml) in RPMI 1640 containing 10% FCS (Biofluids) [or, in some experiments, with 1.5% syngeneic mouse serum (sMS)], 50 µM 2-mercaptoethanol (Sigma), 10 mM HEPES, pH 7.4, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% non-essential amino acids and 1 mM sodium pyruvate (cRPMI), which was supplemented with 10 ng/ml GM-CSF when sMS was used. After 48 h of culture (cLC), non-adherent EC were harvested by vigorous pipetting and LC were enriched by density gradient centrifugation (Lympholyte M). Interphase cells were washed extensively and stained with anti-I-A antibodies for FACS analysis to determine the percentage of LC (which was usually
510% when freshly trypsinized cells were used and 2530% when cLC were used). These cells were used as APC in protein or contact sensitivity assays.
T cell hybridoma
The T cell hybridoma B9.1, specific for the immunodominant peptide HEL103117 (18), was used to detect presentation of HEL by BMDC and LC, and was kindly provided by Dr J. Kanellopoulos (Laboratoire de Biologie Moleculaire du Gene, INSERM U277, Institute Pasteur, Paris, France).
Flow cytometry
Expression of surface molecules was quantified by flow cytometry using the following antibodies: CD4 (H129.19), CD8a (53-6.7), CD11b (M1-70), CD11c (HL3), CD14 (rmC5-3), CD16/32 (2.4G2), CD40 (3/23), CD45R/B220 (RA3-6B2), CD54 (3E2), CD80 (16-10A1), CD86 (GL-1), Gr-1 (RB6-8C5) I-Ad (AMS-32.1), I-Ab (AF6-120.1) and isotype controls, purchased as purified, FITC- or phycoerythrin-conjugated mAb from PharMingen (San Diego, CA). Anti-murine macrophage (F4/80) mAb were purchased from Serotec (Oxford, UK). Staining was performed according to standard techniques and cells were analyzed with a FACScan flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA). Dead cells were excluded from all analysis by propidium iodide (100 nM; Sigma) staining.
In vitro protein antigen presentation assay
To prime T cells with proteins, either 100 µg of HEL or 100 µg of the non-cross reacting protein pigeon CYT (both Sigma) were emulsified in complete Freunds adjuvant (1:1) and injected into the left hind footpads of mice. After 7 days, popliteal lymph nodes were collected, pooled and CD4+ T cells were purified with a mouse CD4 subset column kit (R & D Systems, Minneapolis, MN). In this and all of the other experiments, appropriate sMS was added to the media when T cells were purified for co-culture experiments in order to avoid T cell responses to FCS components. The resulting CD4+ T cell preparations contained <0.2% I-A+ cells. T cells were plated at 2 x 105 cells/well in 96-well flat bottom microplates (Costar, Corning Inc., Corning, NJ) in cRPMI 1640 containing 1.5% heat-inactivated sMS and supplemented with 1 µg/ml indomethacin (Sigma), referred to as proliferation assay medium (PAM). BMDC cultures were fed on day 5 with DCM containing various concentrations of HEL or CYT. DC were purified by metrizamide gradient centrifugation on day 7. The interphase cells were washed extensively and replated at 5 x 105 cell/ml in DCM. On day 8, cells were washed,
-irradiated with 3000 rad and added to T cells to achieve various stimulator:responder ratios. Freshly separated LC were enriched by gradient centrifugation and plated in 10 ml T flasks in complete RPMI containing various concentrations of HEL or CYT. After 48 h of culture, cells were harvested, washed 3 times in HBSS/1.5% sMS,
-irradiated with 1500 rad and added to the T cells. On day 3 of co-culture, [3H]thymidine (Amersham, Biosciences Corp., Piscataway, NJ) (1 µCi/well) was added and T cell proliferation was determined by the incorporation of [3H]thymidine during the last 16 h of culture using a gas ionization counter (Packard, Meriden, CT). Results are presented as the mean (± SEM) of assays performed in triplicates.
Presentation of protein to antigen-specific T cell hybridoma cells in vitro
After incubation of BMDC or LC with HEL or CYT as described above, cells were added to 1 x 105 cells/well of B9.1 T cell hybridoma cells in 96-well flat-bottom microplates and cultured for 24 h at 37°C. One hundred microliters of culture supernatant was removed and assayed for IL-2 content using an ELISA assay (R & D Systems).
In vivo immunization with protein-pulsed BMDC
BMDC were pulsed with 1 mg/ml HEL or CYT as described above. Day 8 cells were washed 3 times with HBSS, and 35 x 105 antigen-pulsed and non-pulsed BMDC were injected into the left hind footpads of naive recipient mice. Seven days after immunization, draining popliteal lymph nodes were harvested and CD4+ T cells were purified as described above. The resulting T cell population contained <0.2 % MHC II+ cells. For assessment of intensity of the T cell responses to the in vivo immunization, LC were pulsed as described above,
-irradiated with 1500 rad, graded numbers of LC were added to 2 x 105 T cells/well and T cell proliferation was assessed as described above. In the cross-presentation experiments, allogenic HEL-pulsed or non-pulsed BMDC were prepared and injected as described above. In the proliferation assay LC syngeneic to the T cells were used as stimulators. The proliferation assays were performed in 1.5% sMS containing PAM. In the cross-presentation experiments using (BALB/c x C57BL/6) F1 mice as hosts, both BALB/c and C57BL/6-derived LC were used as stimulators, and 1.5% F1 sMS was used in the stimulator assays.
Delayed-type hypersensitivity (DTH) assay
To sensitize for protein antigens, mice were injected s.c. at two sites in the lower abdomen either with 25 x 105 protein-pulsed BMDC in 100 µl HBSS solution or, as a positive control, with 100 µl HEL/complete Freunds adjuvant. Non-pulsed BMDC and naive, non-sensitized animals served as negative controls. Challenge and measurement of ear thickness was performed as previously described (19). Each experimental group contained 10 mice.
Hapten modification of cells
Day 8 BMDC and cLC were TNP conjugated as previously described (17). Trypan blue exclusion revealed that 90% of cells were viable.
Sensitization and elicitation of contact hypersensitivity
Graded numbers (104106 cells/mouse) of TNP-modified and unmodified BMDC and LC were injected in 100 µl HBSS/5% FCS s.c. into the dorsal trunk skin of BALB/c, C57/BL6 or F1 mice. As positive controls, mice were sensitized by epicutaneous application of 100 µl 3% trinitrochlorbenzene (TNCB; Polysciences, Warrington, PA) in acetone:olive oil (4:1) to the dry-shaved abdomen. As negative controls mice were painted with the vehicle alone on the abdomen. Challenge and measurement of ear thickness was performed as previously described (17). Experimental groups consisted of five mice each. One-way ANOVA was used to compare experimental and control groups.
Presentation of TNBS by BMDC to TNCB-sensitized CD4+ cells in vitro
Mice were painted with 100 µl of 3% TNCB as previously described (17). Brachial, axillary and inguinal lymph nodes were harvested 6 days later, and purified for CD4+ cells as described above and plated in 96-well plates at 2 x 105 cells in 100 µl/well. BMDC were conjugated with TNBS as previously described, washed extensively and added to wells in various numbers. Proliferation assays were performed as described above.
Assessment of secondary responses of lymph node T cells sensitized by TNBS-conjugated BMDC
BALB/c-derived BMDC were conjugated with TNBS as described above, washed extensively and resuspended in HBSS. TNBS-conjugated or non-conjugated BALB/c-derived BMDC (35 x 105) were injected in 50 µl into the hind footpads of BALB/c and C57BL/6 mice. Draining popliteal lymph nodes were harvested 6 days later and purified for CD4+ cells as in the previous experiments. As stimulator cells, we used BALB/c- and C57BL/6-derived LC, which were cultured in 1.5% sMS-containing medium for 2 days and conjugated with TNBS as described above for BMDC. MHC-matched stimulators and responder cells were co-cultured in the appropriate sMS-containing PAM and proliferation assays were performed.
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Results
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Cell surface phenotype of BMDC generated with GM-CSF and IL-4
Day 8 BMDC cultured with GM-CSF/IL-4 supplemented media expressed high levels of MHC class II molecules, high levels of CD86, CD80 and CD54 co-stimulatory molecules, and were positive for CD11c and CD11b, and negative for CD14 (Fig. 1). Few cells expressed F4/80 molecules. Addition of Escherichia coli lipopolysaccharide (100 ng/ml; provided by Dr Stephanie Vogel, Uniformed Services University of the Health Sciences, Bethesda, MD) or HEL (up to 3 mg/ml) or tumor necrosis factor-
(10 ng/ml; R & D Systems) or anti-CD40 (HM40-3; 10 µg/ml PharMingen) to 8-day gradient-purified BMDC for 2 days did not significantly enhance the surface expression of class II or co-stimulatory molecules (data not shown). These findings suggest that 8-day BMDC, which we used in all of our experiments, exhibited a highly mature phenotype.

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Fig. 1. Cell surface phenotype of BMDC harvested on day 8 cultured with GM-CSF and IL-4. Cells were stained as described in Methods. Cells express high levels of MHC class II molecules, moderate to high levels of CD86, CD80 and CD54 co-stimulatory molecules, were positive for CD11c and CD11b, and negative for CD14. Few cells expressed F4/80 molecules. Shaded areas, mAb of interest; unshaded areas, isotype control mAb. Representative data from one of five experiments.
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BMDC process and present soluble protein antigens in association with MHC II molecules
To assess the in vitro antigen-presenting capacity of BMDC, the cells were either left unpulsed or pulsed with different concentrations (0.13 mg/ml) of HEL or CYT and graded numbers of these cells were added to fixed numbers of HEL- or CYT-primed T cells. BMDC pulsed with 0.1 mg/ml of protein induced little (and in some experiments, no) proliferation of primed T cells. Only BMDC pulsed with high concentrations (0.5 < 1
3 mg/ml) of protein were able to regularly induce significant antigen-specific T cell proliferation (Fig. 2). In subsequent experiments, we used 1 mg/ml protein for pulsing because this concentration consistently induced significant T cell proliferation.

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Fig. 2. High concentration of protein is required to pulse BMDC in order to achieve significant antigen-specific proliferative response of primed T cells in vitro. BMDC were pulsed on day 5 of culture with various concentrations (100 µg, 500 µg, 1 mg and 3 mg) of HEL (BMDC-HEL, closed symbols) or left non-pulsed (BMDC-NP, open symbol). Langerhans cells were pulsed with 100 µg HEL (LC-HEL) or left unpulsed (crossed symbols). On day 7, BMDC were purified by gradient centrifugation and subcultured overnight. Day 8 BMDC and 2-day cultured LC were washed extensively to remove excess antigen, irradiated and co-cultured with 2 x 105 CD4+ T cells purified from draining popliteal lymph nodes 7 days after mice were injected with 100 µg HEL/complete Freunds adjuvant or with 100 µg CYT/complete Freunds adjuvant (hatched symbols) as negative control. The LC number was normalized by the percentage of class II+ cells by flow cytometry in this and all other experiments. Proliferation assays were performed in 1.5% sMS-containing medium in this and all other experiments. When cultured alone, T cells [non-stimulated or staphylococcal enterotoxin B (10 µg/ml) stimulated] as well as irradiated stimulators did not proliferate. In this and all other experiments cell proliferation was measured on day 4 by counting 16 h incorporation of [3H]thymidine (mean ± SE in triplicate wells). Representative data from one of four similar experiments are shown.
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Cultured LC process and present soluble protein antigens and induce enhanced secondary Th cell responses compared to BMDC
When the antigen-presenting capacity of BMDC and LC were compared using different concentrations (0.13 mg/ml) of protein antigen, LC induced highly specific T cell proliferation at lower protein-pulsing concentrations (0.1 mg/ml) (Fig. 2). If, however, higher concentrations were used, BMDC were as potent stimulators as LC, suggesting either that LC might capture or process antigen in a more efficient manner, or that LC express co-stimulatory molecules that function more effectively than those expressed by BMDC. Although this comparison may not provide an important distinction between the two cell types because of the differences in isolation techniques and in culture conditions, the results indicate that the two cell types may differ in their ability to present antigen.
Presentation of HEL to HEL-specific T cell hybridoma cells
To evaluate the capacity of BMDC to present HEL to the HEL-specific T cell hybridoma B9.1, BMDC were pulsed with different concentrations of HEL or CYT (as control antigen) and incubated with the hybridoma cells for 24 h. BMDC pulsed with as little as 10 µg/ml HEL were able to induce specific T cell proliferation as determined by significant IL-2 production (Fig. 3). This result probably reflects the fact that T cell hybridomas require less antigenMHC class II complexes on the APC surface and little or no co-stimulation in order to secrete IL-2.

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Fig. 3. IL-2 production of the HEL-specific T cell hybridoma B9.1 in response to antigen-pulsed BMDC. BMDC were pulsed with various concentrations (1 µg, 10 µg, 100 µg, 1 mg and 3 mg) of HEL (BMDC-HEL) or 3 mg CYT (BMDC-cyto) as described in Fig. 2, harvested on day 8 and washed extensively to remove excess antigen. Irradiated cells were added to 1 x 105 cells/well of B9.1 T cell hybridoma in 96-well flat-bottom microplates and cultured for 24 h at 37°C. Aliquots of 100 µl of culture supernatant were removed and assayed for IL-2 content as a measure of antigen-specific T cell stimulation, using an IL-2 ELISA assay. T cells alone and irradiated BMDC alone did not produce measurable amounts of IL-2. One experiment representative of three is shown.
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Soluble antigen-pulsed BMDC prime lymph node T cells in vivo in an MHC-restricted manner
After pulsing with 1 mg/ml of HEL or CYT, as described above, 35 x 105 BALB/c-derived BMDC were injected into the left hind footpad of BALB/c and C57BL/6 (I-Ad and I-Ab) mice. Draining popliteal lymph nodes were harvested, pooled, and CD4+ T cells were purified and assessed for secondary responses to antigen-pulsed APC. As APC, HEL- or CYT-pulsed cLC were used and added to the syngeneic purified lymph node T cells. Antigen-pulsed LC specifically stimulated those lymph node T cells primed with antigen-pulsed syngeneic BMDC, but not those primed with allogeneic BMDC (Fig. 4). This finding indicates that priming occurs in an MHC-restricted manner and, more importantly, shows that there is no cross-presentation by host APC. It excludes the possibility that there is representation of antigens by autologous APC. To further investigate the phenomenon of cross-presentation on MHC II molecules, we injected 35 x 105 HEL-pulsed and non-pulsed BALB/c- and C57BL/6-derived BMDC into the footpad of F1 (BALB/c x C57BL/6) mice. The draining lymph nodes were harvested 7 days later and CD4+ T cells were purified and assessed for secondary T cell responses as described above. Stimulator HEL-pulsed and non-pulsed LC from BALB/c and C57BL/6 mice were cultured for 2 days in sMS-containing medium, and the proliferation assay was performed in each condition in PAM supplemented with 1.5% serum obtained from F1 mice. BALB/c-derived HEL-pulsed stimulator LC could not induce secondary proliferation of T cells primed with HEL-pulsed C57BL/6 BMDC. In parallel, C57BL/6-derived HEL-pulsed LC did not induce secondary proliferation of T cells harvested from F1 mice primed with BALB/c-derived HEL-pulsed DC (Fig. 5). These findings indicate that peptides presented in association with one of the MHC II type molecules could not induce clonal T cell expansion of T cells with TCR specific for the other MHC class II molecule.

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Fig. 4. Priming for soluble protein antigens occurs in an MHC-restricted manner. Primary sensitization of BALB/c (A) and C57BL/6 (B) mice was attempted by injection of 35 x 105 BALB/c BMDC that had been left unpulsed (open symbols) or were pulsed (closed symbols) with 1 mg/ml HEL as described in Fig. 2 or by injection with 100 µg HEL in complete Freunds adjuvant (hatched symbols). Stimulator cells in the secondary response were unpulsed (squares) or pulsed (triangles) BALB/c- or C57BL/6-derived cultured LC, which were irradiated, washed extensively and plated in 96-well flat-bottom tissue culture plates in graded numbers with 2 x 105 purified BALB/c or C57BL/6 CD4+ lymph node T cells. T cells primed with s.c. injection of syngeneic HEL-pulsed BMDC proliferated in response to syngeneic HEL-pulsed LC, in contrast to T cells primed by allogeneic HEL-pulsed BMDC that did not proliferate. When cultured alone, T cells [non-stimulated or staphylococcal enterotoxin B (10 µg/ml) stimulated] as well as irradiated stimulators did not proliferate. One experiment representative of three is shown.
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Fig. 5. BMDC prime F1 T cells in an MHC II-restricted manner. (A) Primary sensitization of F1 (BALB/c x C57BL/6) mice was attempted by injection of 35 x 105 BALB/c BMDC that had been left unpulsed (open symbols) or were pulsed (closed symbols) with 1 mg/ml HEL as described in Fig. 2. Stimulator cells in the secondary response were unpulsed (squares) or pulsed (diamonds) BALB/c-derived cultured LC and unpulsed (circles) and pulsed (triangles) C57BL/6-derived cultured LC, which were irradiated, washed extensively and plated in 96-well flat-bottom tissue culture plates in graded numbers with 2 x 105 purified F1 CD4+ lymph node T cells. (B) Primary sensitization of F1 mice was attempted by injection of 35 x 105 C57BL/6 BMDC that had been left unpulsed (open symbols) or were pulsed (closed symbols) with 1 mg/ml HEL as described in Fig. 2. Stimulator cells in the secondary response were unpulsed (squares) or pulsed (diamonds) BALB/c-derived cultured LC and unpulsed (circles) or pulsed (triangles) C57BL/6-derived LC, which were irradiated, washed extensively and plated as described in (A). HEL-pulsed LC induced significant secondary proliferation of those F1 T cells primed with syngeneic HEL-pulsed BMDC. In contrast, T cells primed by HEL-pulsed BMDC allogeneic for the stimulators did not proliferate. When cultured alone, T cells [non-stimulated or staphylococcal enterotoxin B (10 µg/ml) stimulated] as well as irradiated stimulators did not proliferate. One experiment representative of three is shown.
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BMDC induce DTH in an MHC-restricted manner
To further evaluate the immunogenic potential of protein-pulsed BMDC in vivo we assessed the mice for expression of DTH. A single s.c. injection of HEL-pulsed BMDC (410 x 105 cells/mouse), but not unpulsed BMDC, led to successful immunization of both BALB/c and C57BL/6 mice. Next we injected HEL-pulsed BALB/c- and C57BL/6-derived BMDC into syngeneic or allogeneic hosts to assess whether allogenic BMDC were able to sensitize mice for HEL. Only when the injected cells were syngeneic to the recipient was a significant foot-swelling response detected. In contrast, if we injected allogeneic HEL-pulsed BMDC, no significant responses were detected (Fig. 6). This in vivo cross-priming experiment demonstrated that soluble antigen-pulsed BMDC cannot immunize allogeneic hosts for protein antigen-specific responses.

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Fig. 6. DTH response of mice immunized with syngeneic or allogeneic HEL-pulsed BMDC. BALB/c- and C57BL/6-derived BMDC were pulsed with 1 mg/ml HEL (BMDC/BA-HEL and BMDC/B6-HEL) as described in Fig. 2 or left unpulsed (BMDC/BA and BMDC/B6). Mice were injected s.c. at two sites in the lower abdominal skin either with 25 x 105 cells in 100 µl HBSS solution or with 100 µl HEL/complete Freunds adjuvant (2 mg HEL/ml) as a positive control. Naive, non-sensitized animals served as negative controls. Six days later mice were challenged by injecting 25 µl of HEL in PBS (2 mg/ml) s.c. into the left footpad, whereas as a control 25 µl of PBS was injected into the right footpad. After 24 h, footpad thickness was measured and the footpad-swelling response was calculated as the thickness of the left footpad minus the swelling of the right footpad. Only syngeneic HEL-pulsed BMDC were able to sensitize for a DTH response; neither non-pulsed nor HEL-pulsed allogeneic BMDC could prime for HEL. Data are representative of three independent experiments, showing the mean ± SEM (n = 10) of the footpad swelling response. *P < 0.05 and **P < 0.01 assessed by one-way ANOVA.
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Hapten-modified BMDC and hapten-modified LC induce comparable amounts of proliferation of antigen-specific T cells
We next determined whether BMDC cultured in this way were able to present haptenated antigens in vitro. We sensitized the mice with 3% TNCB on the abdomen as described above, and harvested the brachial, axillary and inguinal lymph nodes 7 days later. We purified the lymph node cells for CD4+ cells as described above and used them as responders in standard proliferation assays. As stimulators, we used BMDC conjugated or non-conjugated with TNBS. TNBS-conjugated BMDC were able to induce secondary responses in TNCB-sensitized T cells in vitro (Fig. 7). Furthermore, if we compared BMDC to cLC in their ability to present haptens, we found that these two cell types were equally efficient (Fig. 7).

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Fig. 7. In vitro proliferative response of TNCB-sensitized T cells to TNBS-conjugated BMDC and LC. Mice were painted on the shaved thorax and abdomen with 3% TNCB. Cervical, axillary and inguinal lymph nodes were harvested 6 days later, purified for CD4+ T cells and 2 x 105 cells/well of these responder cells were plated in a 96-well flat-bottom tissue culture plate. BMDC and 2-day cultured LC were conjugated with 1 mM TNBS for 10 min (BMDC-TNBS and LC-TNBS), washed extensively, irradiated and plated to achieve various responder:stimulator ratios. Non-conjugated BMDC and LC served as negative controls. TNBS-conjugated BMDC induced a response similar in magnitude to TNBS-conjugated LC. When cultured alone, T cells [non-stimulated or staphylococcal enterotoxin B (10 µg/ml) stimulated] as well as irradiated stimulators did not proliferate. This experiment is representative of two independent experiments.
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Injection of TNP-conjugated DC induces contact hypersensitivity
We next determined whether BMDC were able to present haptenated antigens in vivo. Consistent with previous studies (20,21) we found that naive BALB/c mice injected with graded numbers of TNP-modified or unmodified BMDC developed a contact sensitivity response upon TNCB challenge. Figure 8 shows that as few as 1 x 105 TNP-conjugated BMDC were able to induce a significant contact sensitivity response, comparable in magnitude to the responses generated by an equal number of TNP-conjugated LC. This finding is consistent with the in vitro results showing that there is no significant difference between BMDC and LC in presenting haptens.

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Fig. 8. Injection of TNBS-conjugated BMDC induces contact sensitivity in vivo. BMDC and LC were incubated with 1 mM TNBS for 10 min (LC-TNBS), washed 3 times, injected in 100 µl HBSS/5% FCS s.c. (graded numbers of TNBS conjugated BMDC or 105 LC per mouse) into the dorsal skin of adult BALB/c mice. As a positive control, mice were sensitized by epicutaneous application of 100 µl of 3% TNCB to the abdomen. Mice were painted with the vehicle alone on the abdomen or alternatively injected with non-conjugated BMDC (negative control). Five days later, the right ears were painted with a 1% TNCB solution and ear thickness was measured 24 h later. TNBS-conjugated BMDC (105) were able to induce a significant contact sensitivity response, which was comparable in magnitude to the responses generated by an equal number of TNBS-conjugated LC. Experimental groups consisted of five mice each. *P < 0.05 assessed by two-tailed Students t-test.
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Heat-killed TNP-conjugated BMDC fail to induce contact sensitivity
To further investigate whether BMDC present the haptenated antigen themselves or whether shed antigens may be picked up and presented by host APC, we heat-killed the BMDC either before or after conjugation with TNP. The cells remained morphologically intact and >98% of the cells stained with the vital dye Trypan blue as revealed by microscopic examination. When these heat-killed cells were used there was no significant contact sensitivity response detected. Figure 9 shows that even when large numbers (1 x 107) of TNP-conjugated heat-killed BMDC were used, contact sensitivity responses did not ensue.

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Fig. 9. Heat-killed TNBS-modified BMDC do not sensitize for contact hypersensitivity. BMDC (35 x 105) were injected s.c. as unconjugated cells, TNBS-conjugated live cells (BMDC-TNBS, conjugation as described in Fig. 6.) or heat-killed dead cells (after conjugation cells were cultured at 56°C for 50 min; cell viability assessed by Trypan blue exclusion was <3%). Positive and negative controls were carried out as in Fig. 4. Increase of ear thickness was measured 24 h after challenge. Heat-killed TNBS-conjugated BMDC (107) were not able to induce contact sensitivity. Experimental groups consisted of five mice each. Two-tailed Students t-test was used for statistical analysis.
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TNP-modified BMDC induce contact sensitivity in an MHC-restricted manner as assessed both in vivo and in vitro
To further evaluate whether any cross-presentation occurs during sensitization with BMDC, we harvested BMDC from BALB/c (I-Ad). If we harvested the draining lymph nodes after injecting TNBS-conjugated BALB/c-derived BMDC into BALB/c or C57BL/6 animals and re-stimulated with syngeneic TNBS-conjugated cLC in order to detect secondary T cell responses, we found that there were significant responses only when syngeneic cells were injected. C57BL/6 T cells harvested from mice injected with allogeneic BALB/c-derived TNBS-conjugated BMDC did not proliferate during co-culture with C57BL/6-derived syngeneic TNBS-conjugated LC (Fig. 10).

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Fig. 10. In vitro assessment of T cell responses after in vivo sensitization by TNBS-conjugated syngeneic or allogeneic BMDC. Primary sensitization of BALB/c (A) and C57BL/6 (B) mice was attempted with injection of 35 x 105 BALB/c BMDC that were conjugated with 1 mM TNBS for 10 min, washed extensively (closed symbols) or by painting on the thorax and abdomen with 3% TNCB (hatched symbols). Draining lymph nodes were taken 6 days later, purified for CD4+ T cells, plated in 96-well flat-bottom tissue culture plates (2 x 105 cell/ well) and co-cultured with graded numbers of irradiated syngeneic BALB/c or C57BL/6 2-day cultured LC that were left unpulsed (squares) or were conjugated with TNBS as described in Fig. 6 (triangles). Significant T cell proliferation was detected only when syngeneic but not allogeneic TNBS-conjugated BMDC were used for the primary sensitization. There was no proliferation observed if mice were injected with non-conjugated BMDC or when non-conjugated LC were used as stimulators. When cultured alone, T cells [non-stimulated or staphylococcal enterotoxin B (10 µg/ml) stimulated] as well as irradiated stimulators did not proliferate. Data are representative of two independent experiments.
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Thereafter, we injected the TNBS-conjugated or non-conjugated BALB/c- and C57BL/6-derived BMDC into parent and into F1 mice. Results obtained in three independent experiments (Fig. 11) showed that the TNBS-conjugated BMDC induced contact sensitivity when injected into syngeneic mice and less, but still significant, contact sensitivity in F1 mice. In contrast, if these cells were injected into allogeneic hosts, these mice were unable to develop a contact sensitivity response. Thus, we were able to show, both by in vivo and in vitro assays, that BMDC present haptens and sensitize T cells in an MHC-restricted manner, and that, in keeping with the results of experiments using soluble protein antigens, cross-presentation of haptenated antigens could not be detected.

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Fig. 11. TNBS-conjugated BMDC induce contact sensitivity in an MHC-restricted manner in vivo. BMDC were harvested from BALB/c and C57BL/6 mice, conjugated with 1 mM TNBS for 10 min, washed 3 times, and 35 x 105 of these TNBS-conjugated (BMDC/BA-TNBS, BMDC/B6-TNBS) or non-conjugated BMDC (BMDC/BA, BMDC/B6) were injected s.c. in 100 µl HBSS + 5% FCS into the dorsal area of parenteral and F1 adult mice. Mice were challenged 5 days later as described in Fig. 6 and ear thickness was measured 24 h later. Positive and negative controls were carried out as in Fig. 6. TNBS-conjugated BMDC sensitized syngeneic and, to a lesser extent, F1 mice, but not allogenic mice. Each group contained five mice. The experiment shown is one of three independent experiments. One-way ANOVA was used for statistical analysis.
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Discussion
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In this study, using in vitro and in vivo functional assays we determined the efficiency of BMDC as APC for protein- and hapten-restricted responses. BMDC cultured with GM-CSF and IL-4 for 8 days resembled DC with a highly mature phenotype. If cells were pulsed with soluble protein antigen during maturation, they induced antigen-specific CD4+ T cell proliferation in vitro, suggesting that they effectively processed and presented these antigens. Similarly, BMDC were able to present haptenated antigen to sensitized T cells in vitro, which is consistent with previous findings (17,21,22). We also showed that exogenous proteins and haptens are presented in an MHC-restricted manner; that protein or hapten-pulsed BMDC were not able to immunize allogeneic hosts; and that if parental (A) protein-pulsed BMDC were injected into F1 (A x B) hosts, secondary T cell responses were detected only when T cells were re-stimulated with syngeneic parental (A) antigen-pulsed APC.
These findings demonstrate that haptens and proteins are not cross-presented to CD4+ T cells in vivo, and are in contrast to the phenomenon that has been shown for peptides presented in association with MHC class I molecules. Furthermore, the findings suggest that although uptake of apoptotic (13) and necrotic (15) cells by immature DC has been demonstrated, functional cross-presentation might differ with MHC class II molecules compared to MHC class I molecules. The phenomenon of cross-presentation [see review (23)], when antigens are transferred from cells or tissues and are represented on MHC class I and II molecules of APC, plays an important role in the maintenance of tolerance (2426) and in the rejection of transplants (2730) or tumor cells (31). It is known that immature DC present antigens from endocytosed particles, as shown for bacteria (3234), apoptotic cells (13) and live cells (35). In their work, Albert et al. found that human blood-derived HLA-A2.1+ DC only presented the influenza matrix protein when derived from apoptotic HLA-A2.1 cells; however, in a recent study Inaba et al. showed that processing of cellular fragments onto MHC class II may occur with both apoptotic and necrotic cells (15). Using Y-Ae mAb (36), which recognizes a complex of I-Ab-presenting peptides from another MHC class II product, I-E
, they found large amounts of Y-Ae+ cells in the T cell area of draining lymph nodes of C57BL/6 mice injected with live BALB/c-derived BMDC. These findings indicate that host APC picked up live injected BMDC either in the periphery or in the draining lymph node, therein potentially presenting peptides derived from the injected cells. Inaba et al. (37) have also shown this phenomenon and demonstrated that antigen-laden splenic DC can present antigen efficiently to unseperated lymph node T cells from mice with the same MHC products but not from allogeneic mice. Furthermore, Smith et al. (38) found that despite the fact that peptide-pulsed CD8+ murine splenic DC did not migrate to draining lymph nodes after footpad injection, they were able to stimulate a significant Th cell response.
In our studies we used exogenous proteins and hapten, which were not previously assessed in cross-presentation studies, and our findings indicate that these antigens are not cross-presented to DC 4+ T cells by host APC. Furthermore, in our studies, necrotic (heat-killed) antigen-pulsed cells were not capable of sensitizing the host, even when large numbers were injected, suggesting that although necrotic cells are capable of inducing maturation of APC and of initiating potent immunogenicity of DC, these types of antigens are not represented by the host. This may be due to one of several reasons. It has been shown (39,40) using several transgenic lines expressing different amounts of ovalbumin (OVA) under the control of the rat insulin promoter that the level of antigen expressed by a cell must be above a certain threshold for cross-presentation to occur. It has also been suggested that because a cell expresses enough antigen to be recognized directly by an effector CTL, this does not mean that it will provide sufficient antigen for cross-presentation by professional APC (23). Based on our findings that BMDC required very high levels of soluble antigen to be able to prime naïve T cells or induce antigen-specific secondary T cell proliferation, the antigen presented on the cell surface might still not be sufficient for cross-presentation by host APC.
Recent findings showing cross-presentation of cell-associated OVA required 10 mg/ml of soluble OVA for pulsing spleen cells (41,42), a level significantly higher than the amount we used. In addition, cross-priming was shown to initiate CTL responsesthere is little information about the initiation of CD4 responses. Using OT-I (CD8) and OT-II (CD4) TCR transgenic cell lines it has been demonstrated that cross-presentation of cell-associated OVA required 100-fold more OVA to stimulate CD4+ cells from OT-II mice when compared to CD8+ cells from OT-I mice (42). Although the authors emphasize that there is no way to determine whether the response of OT-I or OT-II cells reflects the strength of the response, or the ability to prime normal naive OVA-specific CD8 or CD4 cells, there may be a difference in the antigenic requirement for cross-presentation of MHC class I- and II-associated molecules. The level of antigen exposure may be increased as the number of APC increases. However, even when we injected as much as 107 cells per animal in order to expose the host to greater amounts of antigen we did not detect cross-presentation to CD4+ T cells or significant DTH or contact sensitivity responses. Taken together, these experiments increase our understanding about the potential in vivo immunogenic effects of BMDC used for vaccination and immunotherapy protocols. They show that, although cross-presentation of cell-associated and soluble antigens has been demonstrated, allogeneic or heat-killed soluble antigen-loaded BMDC are not capable of priming CD4 T cells, indicating that cross-presentation of antigens presented in this way is inefficient.
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Acknowledgements
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The authors thank for Mr Joe Liburd and Ms Michelle Jenkins for their excellent technical support, and Drs Mark Udey, Andrew Blauvelt, Sam Hwang and Akihiko Shibaki for helpful discussions.
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Abbreviations
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APCantigen-presenting cell
BMbone marrow
CTLcytotoxic T lymphocyte
CYTcytochrome c
DCdendritic cell
DCMdendritic cell medium
DTHdelayed-type hypersensitivity
ECepidermal cell
GM-CSFgranulocyte macrophage colony stimulating factor
HELhen egg lysozyme
LCLangerhans cell
OVAovalbumin
PIpropidium iodide
PAMproliferation assay medium
sMSsyngeneic mouse serum
TNCBtrinitrochlorbenzene
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