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*
Department of Microbiology and Immunology and Cancer Center Immunology Program, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and
Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, considerable attention has been focused on the finding that exogenous Ag prepared in particulate form, such as linked to beads, can be internalized by professional APC. The Ags are subsequently processed and the resulting peptides presented in the context of class I MHC (3, 4, 5, 6), thus constituting an exception to the generalization that exogenous Ag is not presented by class I MHC molecules. This phenomenon provides a possible mechanism for stimulation of CD8+ CTL by professional APCs, even if the APC is not synthesizing the antigenic peptides endogenously. One physiologic situation in which this pathway may play a crucial role is in the generation of CTL reactive to tumors. In general, tumor cells fail to express potent costimulatory molecules or stimulatory cytokines, and are therefore inefficient at directly stimulating CTL. Thus, the effective induction of tumor-specific CTL responses probably requires the uptake of exogenous tumor Ag, processing and presentation by professional APCs. Factors that affect these processes, which are thought to be rather inefficient, might significantly enhance the generation of CTL. We have recently shown that IL-3 enhances the generation of exogenous Ag presenting APC and concomitantly enhances the generation of tumor-reactive CTL (7, 8).
These data suggested that IL-3 might be particularly effective in enhancing presentation of exogenous Ag if the Ag could be delivered in a form that was able to be internalized by professional APCs, as the increased number of APCs could then enhance the generation of CTL effector cells. Here we examined whether IL-3 could enhance the presentation of exogenous Ag by APC in vivo and in vitro, and whether cytokine-treated APC charged with Ag could induce a CTL response in naive recipients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybrid (BALB/cByJ x C57BL/6)F1 mice (CByB6F1/J) (H-2d/b), C57BL/6 mice (H-2b), and BALB/cByJ (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). TAP1-deficient mice (9) were generously provided by Dr. Luc Van Kaer (Howard Hughes Medical Research Institute, Vanderbilt University, Nashville, TN). All mice were used at 6 to 8 wk of age.
Cell lines and reagents
Line 1 (L1), a BALB/c (H-2d) alveolar cell lung adenocarcinoma, was originally obtained from Dr. John Yuhas (Oak Ridge National Laboratory, Oak Ridge, TN) (10). IL-3-transfected line 1 (L1/IL-3), which produces 2500 pg/ml IL-3, and IL-4-transfected line 1 (L1/IL-4), which produces 250 ng/ml IL-4, were described previously (7, 11). Line 1/OVA is a stable OVA-expressing line 1 transfectant (8). EL-4 is a thymoma from C57BL/6 (H-2b) mice (12), and EL-4/OVA is a stable OVA-expressing EL-4 line derived by transfection (4). YAC-1 is an A/Sn leukemia (13). B3Z, a T cell hybridoma that recognizes Kb with peptide 257-264 (SIINFEKL) of OVA, was previously described (14). B3Z contains a DNA construct coding for the lacZ gene, under the control of the IL-2 regulatory elements. Upon activation of B3Z through the TCR, the lacZ gene is expressed, allowing determination of the activation of the T cell hybrid through colorimetric assays (14). All cell lines were maintained in vitro in EXCELL-300 (JRH Biosciences, Lenexa, KS) supplemented with 2 or 5% FBS (HyClone, Logan, UT).
Sodium azide, 2-deoxyglucose, cytochalasin B, brefeldin A (BFA), and OVA were obtained from Sigma (St. Louis, MO). Tosyl-activated beads (M-280) were purchased from Dynal (Great Neck, NY). OVA was covalently bound to the beads according to the manufacturer’s instructions and the conjugate was designated as particulate OVA. The BCA protein assay (Pierce, Rockford, IL) was utilized to determine the amount of OVA linked to the beads by the difference in OD between the solution of OVA before and after the conjugation. The concentration of OVA was 80 to 100 µg/mg of beads. IL-3 supernatant and L1 supernatant were prepared as previously described (15). Recombinant mouse IL-3 (rIL-3) was purchased from Collaborative Research Products (Bedford, MA).
In vivo bone marrow and spleen isolation
Tumor cells (105 parental line 1 or L1/IL-3) in a volume of 50 µl were injected i.m. into the left hind thighs of CByB6F1/J mice. Bone marrow and spleen were isolated and dissociated into single-cell suspensions 10 or 20 days later.
In vitro bone marrow culture
Bone marrow cells were isolated from the femurs of naive CByB6F1/J or TAP1-deficient mice, and plated at 2 x 105 cells/well in 12-well plates or 2 x 106 in 100-mm plates. The cells were cultured for 4, 7, and 10 days with recombinant IL-3, L1/IL-3 supernatant, or L1 supernatant as indicated in the appropriate figure legends. Cells were harvested, and tested using the B3Z T hybridoma activation assay.
Generation of L1/OVA tumor cell lysate
L1/OVA (H-2d) tumor cells were treated with DMSO for 7 days to induce class I MHC expression (16). To make allogeneic CTL, each well received 5 x 106 C57BL/6 splenocytes stimulated with 2.5 x 106 irradiated BALB/cByJ splenocytes (2000 rads) in 24-well plates. The final volume for each well was 2 ml. Four days later, cells were harvested and the cytotoxicity was tested against DMSO-induced line 1 (high class I expression). These allogeneic CTL exhibited 65% specific lysis of DMSO-induced line 1 at an E:T ratio of 100:1. To make tumor cell lysate, 1 x 106 DMSO-induced L1/OVA cells were incubated with 1 x 106 allogeneic CTL in 12-well plates for 24 h. Lysates were collected and centrifuged at 1200 x g for 30 min. The supernatant was removed and the pellet was resuspended with 5% FBS-supplemented EXCELL. The Ag concentration of tumor cell lysate as indicated in the figure legends was defined by the number of L1/OVA cells or the concentration of OVA by ELISA as previously described (4).
B3Z T hybridoma activation assays
Activation of B3Z cells was measured by lacZ activity, as previously described (14). Briefly, 5 x 105 putative APCs (as indicated in the appropriate figure legends) and 5 x 105 B3Z cells were mixed in 24-well culture plates, in the presence of varying concentrations of particulate OVA. A total of 16 to 20 h later, the supernatant was removed, the cultures were washed with PBS, and the cells were fixed with cold 2% formaldehyde/0.2% glutaraldehyde for 10 min at 4°C. They were washed again with PBS and overlaid with 250 µl of 0.5 mg/ml X-gal (5-bromo-4-chloro-3-indoyl- ß-D-galactopyranoside; Fisher Biotech, Pittsburgh, PA). The total number of blue cells in the cultures was examined using a microscope after an 8- to 12-h incubation at 37°C.
Inhibition studies of Ag presentation
Naive CByB6F1/J bone marrow-cultured cells treated with or without 250 pg/ml of IL-3 supernatant for 7 days were harvested. In all, 5 x 105 cells/well were plated in 24-well plates in fresh medium overnight before the inhibition assay. In some experiments, cells were incubated with sodium azide (10 mM) and 2-deoxyglucose (60 mM), or cytochalasin B (20 µM) for 1 h at 37°C, and then Ag (tumor cell lysate) was added for another 18 h in the presence of inhibitors. For BFA, bone marrow-cultured cells were first incubated with Ag for 2 h, and then BFA (20 µM) was added for another 17 h in the presence of Ag. Subsequently, cells were fixed with 1% paraformaldehyde, washed twice with medium, and then incubated with 5 x 105 B3Z cells to evaluate Ag presentation.
Priming with particulate Ag-pulsed cells from bone marrow cultures
Bone marrow cells were harvested from femurs of CByB6F1/J mice and plated at 2 x 106 in 100-mm plates for 7 days with 250 pg/ml IL-3 supernatant or control line 1 supernatant. Using L1/OVA tumor cell lysate as exogenous Ag, cells were incubated with lysate prepared from 8 x 106 L1/OVA tumor cells at 37°C for 24 h. After incubation, the cells were collected, washed twice with medium, counted, and injected i.p. into naive CByB6F1/J mice. Additional control animals were injected with L1/OVA tumor cell lysate alone, or were left untreated. Two weeks later, spleens were harvested and 2 x 107 splenocytes were restimulated in vitro with 2 x 107 irradiated EL-4/OVA for 5 days in EXCELL-300 with 5% FBS. Cytotoxicity was measured in a 5-h 51Cr-release assay as previously described by Maryanski et al. (17). Target cells tested were EL-4 and EL-4/OVA.
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Results |
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Our previous work suggested that IL-3 enhances the presentation of exogenous tumor Ags in vivo (8). To further examine this process, we devised a method that delivered the cytokine IL-3 in vivo but enabled us to examine Ag presentation in vitro. IL-3-transfected line 1 (L1/IL-3), the parental line 1 (L1), or control line 1 transfected with IL-4 (L1/IL-4) tumor cells were injected into CByB6F1/J mice (H-2d/b). The tumors were allowed to grow and produce cytokine for 10 or 20 days and then bone marrow and spleen cells were harvested. The efficiency of exogenous Ag presentation with class I MHC was evaluated using a Kb/OVA peptide-specific T cell hybridoma reporter system. B3Z is a T cell hybrid that expresses the lacZ gene upon recognition of an OVA peptide with the class I MHC molecule Kb (14). As the host cells express H-2b, but the line 1 cells, which are H-2d, do not, only host cells can activate the B3Z cells (8). Activated B3Z cells turn blue in the presence of X-gal substrate, allowing simple determination of the number of activated B3Z. For exogenous particulate Ag, we first used OVA-conjugated beads, because this system has been well characterized (3).
Treatment of both bone marrow and spleen cultures with IL-3 resulted in
a population of cells with an enhanced ability to present particulate
OVA (Fig. 1
). The effect on the bone
marrow cells, the more potent of the populations tested, was greatest
at day 10, but also present at day 20. Line 1 cells transfected with
IL-3 also enhanced stimulation of B3Z by spleen cells. Neither the
parental line 1 cells alone nor line 1 cells secreting IL-4 altered the
ability of these cells to present OVA with class I MHC. To eliminate
the possibility that the enhanced Ag presentation of cells by IL-3
resulted from an increased expression of class I MHC, we examined the
class I MHC level of cells by flow cytometric analysis. Both bone
marrow and spleen cells isolated from animals bearing L1/IL-3 tumors
expressed class I MHC molecules at a comparable level to cells from
animals bearing control tumors (data not shown). Taken together, these
results indicate that in vivo exposure to IL-3 by growth of a tumor
secreting the cytokine can increase the ability of APC present in the
bone marrow and spleen to present exogenous particulate Ag with MHC
class I molecules.
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To determine whether our stimulation of APC in vivo by IL-3 could
be replicated in a simpler in vitro environment, bone marrow cells from
unmanipulated mice were cultured in vitro with IL-3 (from L1/IL-3
supernatant), or control line 1 supernatant. After 4, 7, or 10 days,
the cells were harvested, counted, replated at equal cell numbers,
pulsed with particulate OVA, and tested for stimulation of B3Z cells.
Similar to the in vivo data obtained above, cultures of bone marrow
cells treated with IL-3 supernatant in vitro exhibited significantly
higher presentation of particulate OVA than did the control cultures
(Fig. 2A). The increase
in the ability of the cells to present Ag, which was greatest after 7
days in culture, did not appear to be influenced by the dose of IL-3 in
that 62.5 pg/ml was as effective as 250 pg/ml if a constant number of
cells were used (Fig. 2A). However, there was a dose
dependence on the total number of cells recovered from the cultures.
The total number of cells in the IL-3-treated cultures increased four-
to fivefold in number, whereas the control cultures decreased in cell
number by 10-fold (data not shown). The total number of stimulating
cells in the cultures, which takes into account both the increase in
cell number and the enhanced Ag-presenting activity, is shown in Figure 2B and illustrates that the proliferation of the APC was
dependent on the amount of IL-3 used for stimulation. Thus, the higher
doses of IL-3 appear to stimulate the proliferation of both the APC as
well as other cells in the bone marrow cultures. Whereas the ability of
the cells to present particulate OVA was similar at all of the IL-3
doses tested (Fig. 2A), the total yield of APC was
highest at the greatest concentration of IL-3 (Fig. 2B). To ensure that the effects we observed with the
L1/IL-3 were indeed due to IL-3 and not to a combination of IL-3 and
other substances that might be present in the supernatants from the
L1/IL-3 cells, we repeated these experiments using recombinant IL-3 at
the concentration that had given the best response in the initial
experiments (250 pg/ml). Recombinant IL-3 gave very similar results to
L1/IL-3 in terms of the ability of the cells to activate B3Z (Fig. 2C) as well as the total yield of stimulating cells
(Fig. 2D). There was a greater drop in total yield at
day 10 with L1/IL-3 supernatant, probably due to a greater depletion of
media nutrients. Due to the similar effects and the lower cost of
L1/IL-3 supernatant, this reagent was used in all subsequent
experiments. In summary, IL-3 increases both the ability of the cells
to present exogenous particulate Ag in the context of class I and the
total number of cells present. Further, the enhancement by IL-3 of Ag
presentation with class I occurs in vitro as well as following in vivo
exposure.
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To characterize the APC in the bone marrow cultures stimulated in
vitro with IL-3, we determined the cell surface phenotype of the
presenting cells. Because of the heterogeneous nature of the cultures,
this was done by treating the cells with a variety of Abs, followed by
complement treatment and the Ag-presenting activity of the remaining
cells analyzed in the B3Z assay. These results are illustrated in
Figure 3. Abs to CD8, CD4, and B220 all
had little effect, indicating that the APC are neither T nor B cells.
The greatest depletion of activity was seen following treatment with
Abs to the leukocyte common Ag (CD45) and to the macrophage markers
MP20 and Mac-1. The APC activity was also depleted by treatment with
Abs to MHC class II, B7-1, and B7-2. Thus, the cells have the
attributes of professional APC such as macrophages or dendritic cells
(DC). Abs to the macrophage markers F4/80 and FcR also depleted the
activity. Abs to two different DC markers, 33D1 and NLDC, gave somewhat
different results in that anti-NLDC depleted 
45% while the
anti-33D1 depleted only about 30%. These results are perhaps not
surprising given the heterogeneity of the cells within the cultures.
The APC may be macrophages or transitional cells with characteristics
of both DC and macrophages. Alternatively, more than one cell type may
be responsible for the Ag-presenting activity we observe.
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To determine the potential relevance of our observation that IL-3
can enhance exogenous particulate Ag presentation associated with class
I MHC, we evaluated the efficacy of these cells in presenting tumor
fragments/debris. Bone marrow cells from unmanipulated mice were
cultured in vitro with L1/IL-3 supernatant or control line 1
supernatant for 7 days. Cells were harvested and pulsed with titrated
concentrations of L1/OVA tumor cell lysate and tested for stimulation
of B3Z cells. The tumor cell lysate contained no viable cells as
detected by trypan blue exclusion. In all, 108 L1/OVA
lysed cells contained 100 ng of OVA protein as measured by Ab
techniques (ELISA) (4). Figure 4
illustrates the appearance of the APC pulsed with tumor lysate and
mixed with the B3Z cells following development of the assay with the
X-gal substrate. Many more blue, activated B3Z cells were observed in
the wells containing the IL-3-stimulated APC, and the activated T cells
were often in clusters. In contrast, wells with cells from the control
line 1-treated cultures had only an occasional cluster. Our previous
results with this assay (8) have indicated that each of these clusters
usually contains a single APC-like cell. Thus, the frequency of APC
capable of presenting exogenous tumor Ag is enhanced by IL-3. The
number of activated B3Z cells was quantified as illustrated in Figure 5. The cultures of bone marrow cells
treated in vitro with IL-3 were significantly better in presenting
L1/OVA tumor cell lysate than were the control cultures. The small
amount of OVA present within this tumor lysate appears to be very
efficiently presented by the IL-3-stimulated APC.
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To determine whether Ags from the internalized tumor cell lysate
utilized the same class I MHC pathway as endogenously synthesized
proteins, we prepared bone marrow-cultured cells from TAP1-deficient
mice (9) and evaluated their capability to present tumor cell lysate.
Compared with bone marrow cells from wild-type animals, cells from
TAP1-deficient mice were unable to present the tumor cell lysate Ag
(Fig. 6). As a control, cultured bone
marrow cells from TAP1 mutant mice were tested and found to present OVA
peptide, SL8 (SIINFEKL), as well as those from wild-type mice, when SL8
was titrated in the culture medium (data not shown). This observation
suggests that the presentation of tumor cell lysate in the bone marrow
culture system is not simply due to "regurgitation" of peptides
from cytosol into the extracellular environment. Rather, these results
indicate that processing of exogenous tumor cell lysate by the cultured
bone marrow cells uses the classical class I pathway requiring the
TAP1-TAP2 transporter system to transfer the internalized tumor cell
lysate from the cytosol into the ER.
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). The
experiment was repeated twice with similar results. This observation
clearly indicates that energy and phagocytosis are needed for the
presentation of exogenous tumor cell lysate.
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). After removal of
BFA, the B3Z activity was reversed to nearly the same level as that
without the inhibitor, indicating that the BFA effect is reversible
(data not shown). This observation further supports the idea that
presentation of internalized tumor cell lysate requires a functional
secretory pathway. In similar experiments using OVA-conjugated beads as
the Ag, the inhibitors had a similar but stronger effect (over 90%
inhibition) on Ag presentation (data not shown). Taken together, these
results indicate that to present tumor lysate Ag in association with
class I MHC molecules, energy and phagocytosis are required for the
internalization of exogenous Ag. Thus, the mechanism of processing and
presentation by the cultured bone marrow cells appears to be the same
for both forms of Ag. Priming with cells presenting particulate OVA stimulates a potent CTL response in naive recipients
The high efficiency of processing tumor cell lysate and presenting
OVA with MHC class I molecules by naive bone marrow cells stimulated in
vitro with IL-3 led us to examine whether these IL-3-treated cells
could stimulate CTL. Bone marrow cells cultured for 7 days in the
presence of IL-3 or control line 1 supernatant were pulsed with L1/OVA
tumor cell lysate for 24 h. We injected these Ag-charged cells
i.p. into naive mice. Generation of CTL was tested by assaying the
cytotoxicity of restimulated splenocytes against EL-4 and EL-4/OVA
tumor target cells. Mice immunized with the IL-3-treated cells had a
strong specific CTL response against OVA (Fig. 8). In contrast, mice immunized with the
control supernatant-treated bone marrow cells showed no lysis of
EL-4/OVA (Fig. 8). As an additional control, a third group of naive
mice were injected with L1/OVA tumor cell lysate alone at a dose
equivalent to that used to treat the bone marrow cells. No cytotoxicity
response against EL-4/OVA was observed in the spleen cells from these
mice (data not shown). These results demonstrate that the IL-3-treated
cells, following exposure to particulate tumor Ag, can generate a
potent cytotoxic response in naive animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, granulocyte macrophage (GM)-CSF and, as in this study, IL-4,
did not enhance the presentation of exogenous particulate Ag by bone
marrow macrophages (19). In contrast to the cytokines tested in this
earlier study, we have found that IL-3 greatly enhances presentation of
particulate OVA, in both the bead and tumor cell lysate forms. Although
IL-3 has been well documented to stimulate myeloid and other
hemopoietic cell development (20), this paper has shown that IL-3 can
also enhance both the absolute number and frequency of cells capable of
presenting exogenous Ag in a class I-restricted fashion. The experiments presented here suggest that the percentage of APC capable of presenting either bead or tumor cell lysate is extremely low in untreated bone marrow. Interestingly, Reis e Sousa and Germain also found that only a small portion of Ag-presenting thioglycolate-induced macrophages were susceptible to lysis by CTL, implying that only a subset of cells was presenting antigenic peptide with class I MHC (21). In a similar vein, Norbury et al. observed that a small percentage of their bone marrow macrophages could present exogenous Ag to the B3Z hybridoma (22). After treatment with IL-3, we found that the presenting ability of these bone marrow APC increased by over 50-fold, and they displayed CTL-priming ability whereas bone marrow cells that were not treated with IL-3 were not able to prime T cells effectively. Although it remains formally possible that some of the increase is due to better Ag presentation by the same cell, given the proliferation observed in the IL-3-treated cultures and the direct evidence of increased numbers of clusters, we think the simplest and most conservative interpretation is that the cells with this activity are simply more frequent. Taken together, these observations suggest that the APC that can efficiently present exogenous Ag may be rare. Consequently, the number of these APC may be a limiting step in determining the magnitude of antitumor CTL responses. Thus, cytokines that enhance the generation of APC capable of representing exogenous Ag could have a pivotal role in determining the antitumor CTL response to tumors (23).
To characterize the phenotype of the IL-3-treated bone marrow APC responsible for exogenous Ag presentation, we used Ab and complement depletion studies, combined with functional studies of Ag presentation. The presenting bone marrow cells have many of the expected properties of professional APC, including expression of leukocyte common antigen (LCA), class II Ags, and costimulatory molecules such as B7-1 and B7-2. Interestingly, however, the results with macrophage and DC markers did not clearly delineate these lineages. One possibility is that the IL-3-treated bone marrow cultures contain a heterogeneous population of APC, possibly containing both macrophages and DC, each with exogenous APC ability. Alternatively, these results may reflect a single cell with properties of both cell types. In this regard, some recent reports have indicated that the traditional criteria used to distinguish macrophages and DC, including stimulation of MLR, morphology, surface markers, adherence ability, and phagocytic function, may not hold, especially during the immature or precursor stages (24, 25). For instance, DC progenitors have been shown to phagocytose particulate Ag containing Bacillus Calmette-Guérin organisms (26); likewise, monocyte-derived macrophages have been reported to stimulate an allogeneic MLR (27). In addition, Szabolcs et al. have shown that both macrophages and DCs can be stimulated from a common progenitor cell in human bone marrow (28). Therefore, according to the developmental plasticity of the myeloid lineage, our IL-3 treatment of bone marrow APC revealing cells with both macrophage and DC traits is not without precedent (24, 25).
Presentation of exogenous Ags with class I MHC was first demonstrated
by Bevan (29) for minor histocompatibility Ags, Gordon et al. (30) for
H-Y Ags, and Gooding and Edwards (31) for SV40 Ags in vivo. More recent
studies have extended this concept and utilized particulate Ags such as
latex beads to demonstrate this alternative class I MHC presentation
(32). In agreement with other reports (3, 33), the bone marrow-derived
APC described here were able to efficiently process and present
exogenous Ag in the context of class I MHC, when the Ag was conjugated
to beads. Importantly, when pulsed with lysate from killed
Ag-expressing tumor cells, a form of Ag that might be encountered in
vivo, the IL-3-cultured bone marrow cells presented the Ag very
effectively. Although it is difficult to precisely determine the amount
of Ag in these preparations, based on an ELISA determination, the
concentration of OVA present in the tumor lysate is low (1
ng/106 cells). However, it should be noted that this may be
an underestimate of the total amount of OVA since the antigenic
determinants recognized by the Abs used in the ELISA assay are probably
different from the antigenic peptides recognized by the T cells, and
some of the Ab epitopes likely depend on conformation of the molecule.
Nevertheless, these estimates suggest that the process of
representation in these cells is remarkably efficient. Interestingly,
Ags such as those complexed with tumor-derived heat shock proteins have
been reported to be highly antigenic in vivo (34). Further, the use of
cell debris or irradiated whole mammalian cells as exogenous Ags to
prime CTL responses in vivo has been reported (35, 36), and has been
used for a number of years in tumor immunology. Debrick, Campbell, and
Staerz found that mice immunized i.v. with irradiated, OVA-expressing
EL-4 tumor cells or sonicated equivalents could induce OVA-specific CTL
(26). These experiments also suggested that macrophages may act as
accessory cells for this class I MHC-restricted immune response (26).
Carbone and Bevan also reported that the transfer of irradiated
OVA-expressing, syngeneic splenocytes into naive recipients can result
in the development of CTL immunity (35). The cells obtained from the
IL-3-treated bone marrow cultures may be very useful in further
characterizing how this process occurs.
Using the bone marrow from TAP1/2 knockout mice and inhibitors, the
presentation of Ag-expressing tumor cell lysate by IL-3-treated, bone
marrow-derived APC was found to require energy, phagocytosis, and
access to the class I MHC pathway to stimulate CD8+ T cells
(Fig. 7). This linking of the phagosome-to-cytosol pathway with the
conventional class I MHC pathway has been reported by
Kovacsovics-Bankwoski and Rock using OVA-conjugated latex beads as Ag
and peritoneal and bone marrow macrophages as APC (36). Supporting
these previous results, we performed similar experiments, using the
Ag-coupled beads as an exogenous Ag. An identical inhibition pattern
was found (K. Yeh, J. Frelinger, and E. Lord, unpublished
observations). Furthermore, these results suggest that the same
mechanisms for Ag internalization and processing are used for both the
Ag-expressing tumor cell lysate and the Ag-coupled beads.
An interesting issue is what might initiate this process of tumor
fragmentation in vivo. Such fragments might originate from a direct
attack of the tumor by nonspecific cells of the immune system such as
macrophages, granulocytes, or NK cells. This would be consistent with
reports demonstrating a link between the innate and the adaptive immune
response to tumors (37). Alternatively, physical stresses, such as
insufficient vascularization resulting in nutrient or oxygen
deprivation, could also result in apoptosis and subsequent release of
tumor fragments. In either scenario, host APC could then take up these
tumor fragments and re-present tumor Ag to CTL precursors. In this
regard, we have shown that IL-3-treated bone marrow APC internalize
Ag-expressing tumor cell lysate, and engender a potent CTL response
when transferred to naive recipients (Fig. 8). Thus, presentation of
exogenous Ag by bone marrow APC may be very relevant to the in vivo
situation. Interestingly, GM-CSF, a cytokine that is functionally
related to IL-3 though generally thought to act on a more
differentiated population in hemopoiesis, has been used as an adjuvant
to elicit CTL responses in some tumor models (38). In mouse chimera
studies, GM-CSF was found to promote the differentiation of host APC
derived from the bone marrow, leading to tumor-reactive CTL generation
that could only arise from re-presentation (39). Our previous data that
IL-3 enhances CTL development, via the increased presentation of tumor
Ag by rare host APC (8), also suggest that host APC can play a
significant role in the generation of tumor-reactive CTL. Understanding
the precise mechanisms of how cytokines such as GM-CSF and IL-3
function will be important in effectively using them as adjuvants
in cancer immunotherapy.
In summary, this paper has clearly demonstrated that IL-3-treated bone marrow cells can act as potent APC for presenting tumor Ags to CTL. This has profound implications for the development of new methods to enhance the generation of tumor-reactive immune cells. IL-3 may be just one of a cascade of cytokines needed to generate both specific CTL and helper cells as well as nonspecific effector cells. It is possible that tumor cell lysates could be used in conjunction with IL-3 and GM-CSF to provide maximal stimulation of APC capable of optimally presenting Ag to a variety of T cell types. Such APC might also produce additional cytokines such as IL-12 to further enhance antitumor immunity. If, as our data suggest, APC are the limiting cells in this process, methods such as those suggested in this study have considerable potential for enhancing immunotherapy, even in tumors in which the tumor Ag has not yet been identified.
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Footnotes |
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2 A. J. McAdam was supported in part by Grant T32GM07356 from the National Institutes of Health. Current address: Department of Pathology, Brigham and Woman’s Hospital, 75 Francis Street, Boston, MA 02115.
3 B. A. Pulaski was supported in part by Grant T32AI07285 from the National Institutes of Health. Current address: Department of Biologic Science, University of Maryland, Baltimore, MD.
4 Address correspondence and reprint requests to Dr. Edith M. Lord, University of Rochester, School of Medicine and Dentistry, Box 704, 601 Elmwood Avenue, Rochester, New York 14642.
5 Abbreviations used in this paper: ER, endoplasmic reticulum; B3Z, B3Z86/90.14; TAP, transporters associated with antigen presentation; BFA, brefeldin A; DC, dendritic cell; GM, granulocyte macrophage.
Received for publication April 21, 1997. Accepted for publication February 11, 1998.
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