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Immunology |
Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-1750 [M.Z., H.T., J.G., P.A., J.S., K.Y.T.], and Therion Biologics Corporation, Cambridge, Massachusetts 02142 [D.P.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DCs are the most potent APCs, partly because they express higher levels of MHC molecules and a variety of costimulatory molecules. Previous studies (6, 7, 8)
have demonstrated a rather homogeneous expression of costimulatory molecules on murine DCs. Murine DCs are usually generated from bone marrow and cultured with GM-CSF and IL-4. As defined by the phenotypic profile, the DCs described were mature DCs (high expression of CD80, ICAM-1, and LFA-3). Previous studies (9, 10, 11, 12, 13, 14, 15, 16, 17)
also have shown a considerable variability in the levels of costimulatory molecule expression on human DCs prepared from human PBMCs. This variation is seen in DCs derived from different donors, regardless of what DC preparation methods are used. Previous studies have shown B7-1 to be expressed on 2–60% of DCs, depending on donors, when DCs were prepared from human PBMCs and then cultured with GM-CSF and IL-4. This variation has also been seen in more mature DCs additionally treated with TNF-
and/or CD40L (18, 19, 20, 21)
. Nonetheless, the degree of efficacy of a DC in activating T cells seems to be related, at least in part, to the degree and level of expression of certain costimulatory molecules on the DCs.
Strategies are being devised in which DCs can be engineered to hyperexpress costimulatory molecules and, thus, potentially increase their efficacy in T-cell activation. Recent studies have demonstrated that poxvirus recombinants can be constructed to express a triad of murine costimulatory molecules (22) . Poxvirus vectors were used because of their ability to insert large amounts of foreign genes and multiple transgenes (23 , 24) . The two poxvirus vectors used were the replication-defective fowlpox (avipox) virus and the replicationcompetent vaccinia virus. Fowlpox is replication-competent in avian cells; it can infect mammalian cells and express transgene products via early viral promoters, but it will not express many viral structural proteins and will not replicate. The rF that was used in previous studies was shown to efficiently express murine B7-1, murine ICAM-1, and murine LFA-3 transgenes, and was designated rF-TRICOM (mu). The acronym TRICOM was used to denote a triad of costimulatory molecules. Studies with this vector and with rV-TRICOM (mu) demonstrated that enhanced activation of T cells could be achieved upon infection of a murine tumor cell when using either Concanavalin A or peptide as signal 1. Recent studies demonstrated that infection of murine DCs with rF-TRICOM or rV-TRICOM led to enhanced levels of expression of the TRICOM and enhanced activation of murine naïve and effector T cells, using Concanavalin A and OVA peptide as signal 1, respectively (22) .
Avipox recombinant vectors containing transgenes for viral proteins or TAAs have now been used in numerous clinical trials and have been shown to be safe and capable of inducing specific human T-cell responses. Recent studies have also demonstrated that if avipox recombinants containing the human CEA transgene are given numerous times to patients with advanced cancer, increases in specific T-cell responses to the CEA transgene may occur (25, 26, 27) . Human DCs, moreover, are now being used in numerous clinical trials for a range of human cancers (28 , 29) . These trials are using DCs of various levels of maturity. In the studies reported here, we describe an avipox vector that contains the transgenes for three different human costimulatory molecules: B7-1, ICAM-1, and LFA-3, designated rF-TRICOM. The homologies between murine and human CD80, ICAM-1, and LFA-3 are 44%, 50%, and 25% at amino acid level, respectively (30, 31, 32) . We report here that human DC populations can be efficiently infected with this vector and, consequently, hyperexpress all three costimulatory molecules. Using PBMCs from several apparently healthy donors as a source of DCs and T cells, and using a Flu 9-mer peptide to provide signal 1, we demonstrate that infection of human DCs with rF-TRICOM can greatly enhance the level of T-cell activation at different concentrations of signal 1. In addition, far fewer DCs are required to activate T cells when DCs infected with TRICOM are used, no increase in T-cell apoptosis is seen when T cells are activated to higher levels by rF-TRICOM-infected DCs, and rF-TRICOM can enhance the ability of DCs of different maturity levels to activate T cells. The phenomenon of enhanced activation of naïve T cells by rF-TRICOM-infected DCs was also observed by using, as signal 1, a peptide of HPV, which is associated with numerous cancers, and the immunodominant peptides of the self-antigens CEA and PSA. None of the studies showed any effects with the use of control fowlpox-wild type vector (FP-WT) alone. The results reported here thus demonstrated that the infection of human DCs with a vector capable of hyperexpressing three human costimulatory molecules could activate T cells to a new threshold of activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Culture of DCs from PBMCs
All experiments involving patient material were conducted according to NIH guidelines, and all patients provided written, informed consent. PBMCs from apparently healthy donors were separated using lymphocyte separation medium gradient (Organon Teknika, Durham, NC), as described previously (37)
. DCs were prepared using a modification of the procedure described by Sallusto et al. (38)
. PBMCs (1.5 x 108) were resuspended in AIM-V medium containing 2 mM glutamine, 50 µg/ml streptomycin, and 10 µg/ml gentamicin (Life Technologies, Inc.) and allowed to adhere to a T-150 flask (Corning Costar Corp., Cambridge, MA). After 2 h at 37°C, the nonadherent cells were removed with a gentle rinse. The adherent cells were cultured for 6–7 days in AIM-V medium containing 100 ng/ml of rhGM-CSF (Pepro Tech, Inc., Rocky Hill, NJ) and 20 ng/ml of rhIL-4 (Pepro Tech, Inc.). The culture medium was replenished every 3 days. rhTNF- (Pepro Tech, Inc.) was added into the culture medium at a concentration of 20 ng/ml, in addition to GM-CSF and IL-4, for the experiments in which rhTNF- was used for the DC maturation. For CD40L treatment, CD40L (Pepro Tech, Inc.) was added to the uninfected DC cultures at a concentration of 2 µg/ml on day 6 for 24 h, and the CD40L-treated DCs were used as APCs.
Recombinant Viruses
The recombinant fowlpox virus rF-TRICOM contains the genes for the human costimulatory molecules CD80, ICAM-1, and LFA-3. rF-TRICOM was constructed by the insertion of foreign sequences into the BamHI J region of the genome of the POXVAC-TC strain of fowlpox virus using methods as described (39)
. rF-TRICOM contains the human LFA-3 gene under the control of the vaccinia 30K (M2L) promoter, the human ICAM-1 gene under control of the vaccinia I3 promoter (40)
, and the human B7-1 gene under the control of the synthetic E/L promoter (41)
. rF-TRICOM also contains the lacZ gene under the control of the fowlpox C1 promoter (39)
. All studies used FP-WT as a control vector.
Infection of DCs
DCs (1 x 106) were incubated in 1 ml of Opti-MEM medium (Life Technologies, Inc.) at 37°C with FP-WT or rF-TRICOM. Titration experiments demonstrated that 4 x 107 plaque-forming units/ml, equal to an MOI of 40:1 for 2 h, were able to consistently induce transgenes expression in 
75% of the infected DCs. The infected DCs were suspended in 10 ml of fresh, warm CM containing 100 ng/ml of rhGM-CSF and 20 ng/ml of rhIL-4, cultured for 24 h, and then subsequently used as stimulators.
Peptide
The following HLA-A2 binding peptides were used to pulse DCs: (a) CAP1–6D (42
, 43)
, CEA amino acid position 571–579 YLSGADLNL, designated CEA peptide; (b) influenza matrix protein peptide 58–66 GILGFVFTL, designated Flu peptide (44)
; (c) HPV type 16 E7 peptide 11–20 YMLDLQPETT (45)
; and (d) PSA peptide (PSA-3) 154–163 VISNDVCAQV (46)
. All peptides were synthesized by Multiple Peptide Systems (San Diego, CA), and their purity was >96%.
Generation of T-cell Lines
Modification of the protocol described by Tsang et al. (35)
was used to generate peptide-specific CTLs. Uninfected DCs and DCs infected with rF-TRICOM or control vector FP-WT were used as APCs. CEA, HPV, and PSA peptides were added to the uninfected or infected DCs at a final concentration of 25–50 µg/ml, and Flu peptide was added at a concentration of 0.02 µg/ml. Autologous nonadherent cells from PBMCs were added to APCs at an APC:effector cell ratio of 1:10. Cultures were then incubated for 3 days at 37°C in a humidified atmosphere containing 5% CO2. After removal of the peptide-containing medium, the cultures were supplemented with IL-2 at a concentration of 20 units/ml for 7 days, with IL-2-containing medium replenished every 3 days. The 3-day incubation with peptide and 7-day IL-2 supplement constituted one IVS cycle. Primary cultures were restimulated with specific peptide on day 11 to begin the next IVS cycle. Irradiated (23,000 rads), autologous EBV-transformed B cells were used as APCs beginning with IVS-3.
Flow Cytometry
Single-color Flow Cytometric Analysis.
The method for single-color flow cytometric analysis has been described previously (47)
. Briefly, cells were washed three times with cold Ca2+ and Mg2+-free Dulbecco’s phosphate-buffered saline and then stained for 1 h with monoclonal Abs against HLA-A2 (A2, 69; One Lambda, Inc., Canoga Park, CA) using 10 µl of the 1x working dilution/106 cells. Mineral oil plasmacytoma-104E (Cappel/Organon Teknika Corp., West Chester, PA) was used as an isotype control. Then the cells were washed three times and incubated with a 1:100 dilution of FITC-labeled goat antimouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The cells were analyzed immediately using a Becton Dickinson FACScan equipped with a blue laser with an excitation of 15 nW at 488 nm. Data were gathered from 10,000 live cells, stored, and used to generate results.
Dual-color Flow Cytometric Analysis.
The procedure for dual-color flow cytometric analysis was similar to that for single-color analysis with the following exceptions. The Abs used were anti-CD80 FITC/anti-CD86 PE, anti-CD58 FITC/anti-CD54 PE, anti-CD3 FITC/anti-CD14 PE, anti-class II FITC/anti-CD11c PE, anti-CD83 FITC/anti-class I PE, and anti-IgG1 FITC/anti-IgG2a PE (isotype controls). All of the Abs were purchased from Becton Dickinson (BD/PharMingen, San Diego, CA). Staining was done simultaneously for 1 h, after which cells were washed three times, resuspended as above and immediately analyzed using a Becton Dickinson FACScan equipped with a blue laser with an excitation of 15 nW at 488 nm, with the use of the CELLQuest program.
Cytotoxic Assay
Target cells were labeled with 50 µCi of 111Indium-labeled oxyquinoline (Medi-Physics Inc., Arlington, IL) for 15 min at room temperature. Target cells (0.3 x 104) in 100 µl of RPMI 1640 CM were added to each well of 96-well flat-bottomed assay plates (Corning Costar Corp.). The labeled target cells were incubated with peptides for 60 min at 37°C in 5% CO2 before effector cells were added. Effector cells were suspended in 100 µl of CM supplemented with 10% pooled human AB serum and added to the target cells. The plates were then incubated at 37°C in 5% CO2 for 4 or 16 h. Supernatant was harvested for gamma counting with harvester frames (Skatron, Inc., Sterling, VA). Determinations were carried out in triplicate, and SDs were calculated. Specific lysis was calculated with the following formula (all values in cpm):
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Spontaneous release was determined from wells to which 100 µl of CM were added. Total releasable radioactivity was obtained after treatment of targets with 2.5% Triton X-100.
HLA Typing
HLA phenotyping was performed by the Blood Bank of the NIH using a standard Ab-dependent microcytotoxicity assay and a defined panel of anti-HLA antisera. All donors were HLA-A2 positive. The class I phenotype of the V8T cell line was HLA-A2, -; B18 (W6), 44 (12, W4).
Cytokine Detection
Supernatant of T cells, exposed for 24 h to infected or uninfected DCs pulsed with peptide in IL-2-free medium at various responder:stimulator ratios, were screened for secretion of IFN-
using an ELISA kit (R & D Systems, Minneapolis, MN). IL-12 p70 determination was also performed using an ELISA kit (R & D Systems). The results were expressed in pg/ml.
Apoptosis Assay
T cells were incubated for 48 h in the presence of peptide-pulsed DCs, as described in the IVS procedure, and replated to 96-well plates for 24 h. Apoptosis was analyzed using the TUNEL assay (48)
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Statistical Analysis
Statistical analysis of differences between means was performed using a two-tailed t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2–4% of the starting PBMC population. FACS analysis after culture in the presence of rhGM-CSF and rhIL-4 (see "Materials and Methods") revealed a considerable variation among donors in the level of costimulatory molecules expressed on the surface of DCs before infection with rF-TRICOM. B7-1 expression ranged from 5.3% to 23.5% of cells, which is similar to the range reported previously by several groups (9, 10, 11, 12, 13, 14, 16, 17)
. ICAM-1 and LFA-3 were expressed on 50% and 85% of DCs, respectively. Compared with uninfected DCs (Fig. 1A)
, rF-TRICOM-infected DCs showed increases both in the percentage of cells expressing B7-1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58) and in the expression level of each of these molecules, as determined by the MFI (Fig. 1C)
. Infection of DCs with FP-WT control vector had no effect on the expression of any of the three costimulatory molecules (Fig. 1B)
. Expression of other markers (CD86, CD40, Class II, CD11c, CD3) used to phenotype DCs, however, was not affected by either FP-WT or rF-TRICOM infection (Fig. 1, B and C)
. Infection of DCs with rF-TRICOM at different MOIs revealed that an MOI of 40 was optimal in terms of increasing both the percentage of positive cells and the MFI for B7-1, ICAM-1 and LFA-3 expression on DCs.
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production after 24 h of incubation. As seen in Table 1
, the use of control DCs (i.e., uninfected DCs) pulsed with Flu peptide as APCs resulted in activation of T cells from PBMCs of one of the three donors (donor 3). A similar level of activation for this donor was seen using DCs infected with FP-WT. Infection of DCs with the rF-TRICOM vector in the absence of Flu peptide did not change T-cell stimulation in any of the 3 donors. However, infection of peptide-pulsed DCs with rF-TRICOM led to substantial increases in IFN- production in all three donors (Table 1)
. To study this phenomenon further, DCs pulsed with different concentrations of Flu peptide were used. At each concentration of peptide, rF-TRICOM infection of DCs led to increases in IFN- production by activated T cells (Fig. 2A)
. Infection of peptide-pulsed DCs with FP-WT yielded results identical to those seen with uninfected DCs. To quantitate this phenomenon further, different effector cell:DC (APC) ratios were used. As can be seen in Fig. 2B
, little if any IFN- production was seen using DCs from this donor at the various effector cell:APC ratios. Substantial increases in IFN- production by activated T cells were seen, however, when peptide-pulsed DCs were infected with rF-TRICOM at all effector cell:APC ratios, even at a ratio of 200:1 (Fig. 2B)
. Again, infection of DCs with FP-WT had no effect.
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, as well as rhGM-CSF and rhIL-4, for 7 days (see "Materials and Methods"). As seen in Table 2
, additionally maturing DCs with rhTNF- led to a substantial increase in CD80 expression, as well as some increase in CD54 and CD83 expression. In comparison, infection of DCs (generated with rhGM-CSF and rhIL-4) with rF-TRICOM resulted in a phenotype with additionally increased expression of CD80, CD54, and CD58 (Table 2)
. Using the DCs matured with rhGM-CSF, rhIL-4, and rhTNF-, IFN- release was observed when pulsing with increasing concentrations of Flu peptide. Infection of peptide-pulsed DCs (generated with rhGM-CSF and rhIL-4) with rF-TRICOM, however, resulted in enhanced IFN- production by activated T cells at every level of peptide used for signal 1 (Fig. 2C)
. Infection of these DCs with FP-WT had no enhancing effect on IFN- production at all peptide concentrations. Subsequent to treatment with rhGM-CSF, rhIL-4, and rhTNF-, DCs were matured further with CD40L. This led to increased expression of CD83, CD80, and CD86 (Table 3)
. DCs from this same donor, which had been treated with GM-CSF, IL-4, and TNF-, were also infected with rF-TRICOM and phenotyped. This led to increased expression of CD80, CD54, and CD58, as expected. When these different DC preparations from the same donor were pulsed with 0.5 µg/ml Flu peptide and incubated with the donor’s T cells (at an effector cell:APC ratio of 10:1) for 24 h, only the peptide-pulsed DCs infected with rF-TRICOM produced substantial levels of IFN- (Table 3)
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, stimulation of T cells with rF-TRICOM-infected peptide-pulsed DCs did not lead to increases in T-cell apoptosis. Indeed, slightly lower levels of apoptosis (5.5%) were seen in T cells stimulated with peptide-pulsed, rF-TRICOM-infected DCs (Fig. 3D)
, as compared with T cells stimulated with peptide-pulsed, uninfected DCs or with FP-WT-infected DCs (7.9 and 12.7% apoptosis, respectively; Fig. 3, B
and C).
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To examine further the biological activity of T cells activated with peptide-pulsed, rF-TRICOM-infected DCs, Flu peptide-specific T-cell lines were established using (a) peptide-pulsed DCs; (b) peptide-pulsed DCs infected with FP-WT; or (c) peptide-pulsed DCs infected with rF-TRICOM as APCs. T cells were used at IVS-8 to determine CTL activity. C1R-A2 cells with or without Flu peptide were used as targets. As can be seen in Table 4
, the extent of lysis of peptide-pulsed targets observed was greater when using rF-TRICOM-infected, peptide-pulsed DCs as APCs as compared with the lysis observed with T cells derived from either uninfected DCs or fowlpox-infected DCs pulsed with peptide. Although this increase in lysis simply may be attributed to increased numbers of peptide-specific T cells in lines established with rF-TRICOM-infected peptide-pulsed DCs as APCs, it does demonstrate the generation of a functional T-cell capable of lysis when this type of APC is used.
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. When compared with control Ab, the treatment of peptide-pulsed, rF-TRICOM-infected DCs with anti-CD80, anti-CD54, or anti-CD58 Abs clearly reduced the level of IFN- production by T cells. The combined use of any two of these Abs reduced further the level of IFN- production by T cells, and the use of all three of these Abs completely eliminated IFN- production (Table 5)
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, the use of control uninfected DCs or DCs infected with FP-WT, with or without HPV peptide, resulted in little, if any, IFN- production by T cells obtained from PBMCs from both donors. Similarly, the use of rF-TRICOM-infected DCs without peptide as APCs did not result in IFN- production. However, the use of peptide-pulsed, rF-TRICOM-infected DCs did result in IFN- production by autologous T cells from PBMCs from donors 1 and 2, albeit at low levels (Table 1)
. When T cells from donor 2 were stimulated in culture with HPV peptide through an additional IVS cycle, similar trends were noted. Using these T cells, no IFN- production was noted when DCs devoid of peptide were used, whether or not they were infected with vectors or uninfected. The use of peptide-pulsed DCs infected with FP-WT control vector somewhat increased the level of IFN- by T cells. However, far greater levels of IFN- production by T cells were noted when peptide-pulsed, rF-TRICOM-infected DCs were used as APCs (Table 1)
. The effect of different peptide concentrations on enhanced T-cell activation using rF-TRICOM-infected DCs was then analyzed. At each HPV peptide concentration, marked enhancement of T-cell activity was seen using rF-TRICOM-infected DCs as APCs, as compared with using uninfected DCs or FP-WT-infected DCs (Fig. 4A)
. Indeed, 10-fold less HPV peptide was required to activate T cells to produce a given level of IFN- when using rF-TRICOM-infected DCs, as compared with using uninfected DCs or DCs infected with the control vector FP-WT. These results were quantitated further using different effector-cell:DC ratios. As seen in Fig. 4B
, peptide-pulsed DCs, either uninfected or infected with FP-WT, increased IFN- production at effector cell:DC ratios of 50:1 and 10:1. At all effector cell:DC ratios, however, rF-TRICOM-infected, peptide-pulsed DCs used as APCs demonstrated substantial increases in T-cell activation. These results demonstrated that similar levels of IFN- production by T cells can be achieved by using 10-fold fewer peptide-pulsed, TRICOM-infected DCs as APCs, as compared with using uninfected, peptide-pulsed DCs as APCs.
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, T-cell lines generated by HPV peptide-pulsed DCs infected with TRICOM had the ability to lyse HPV peptide-pulsed targets to a degree greater than that observed when using T cells generated with either peptide-pulsed uninfected DCs or peptide-pulsed, FP-WT-infected DCs.
The above results demonstrated that infection of peptide-pulsed DCs with rF-TRICOM could enhance the activation of T cells to epitopes of viral proteins. Studies were also undertaken to determine whether this enhanced T-cell activation by rF-TRICOM-infected DCs could be achieved using peptides to two different human "self" TAAs: the PSA-3 peptide of PSA and the 9-mer agonist epitope CAP-1-6D of human CEA. The characteristics of the CEA-specific T-cell line V8T have been described (35
, 36)
. CEA peptide-pulsed, uninfected DCs or peptide-pulsed DCs infected with FP-WT were able to stimulate the CEA-specific T-cell line to produce 310 and 226 pg/ml of IFN-, respectively, whereas no stimulation was seen in the absence of peptide (<50 pg/ml of IFN- produced). Enhanced levels of T-cell stimulation (604 pg/ml of IFN- produced), however, were observed when CEA peptide-pulsed DCs infected with rF-TRICOM were used as APCs.
Studies were then conducted to determine whether the use of rF-TRICOM-infected, peptide-pulsed DCs as APCs could aid in the generation of T cells specific for the PSA epitope PSA-3. DCs derived from a healthy individual who has the HLA-A2 allele were cultured in rhGM-CSF and rhIL-4 for 7 days. DCs were pulsed with 50 µg/ml of PSA-3 peptide and used at an effector cell:APC ratio of 10:1. IFN- production was induced in T cells from this donor in response to the PSA-3 peptide-pulsed DCs only after three IVS cycles; higher levels of IFN- (2500 pg/ml) were produced from T cells stimulated with PSA peptide-pulsed DCs infected with TRICOM as APCs, as compared with the peptide-pulsed, uninfected DCs or peptide-pulsed, FP-WT-infected DCs (1000 pg/ml). To quantitate this phenomenon, these T cells were exposed to autologous DCs pulsed with different concentrations of PSA peptide. As seen in Fig. 5A
, using these lower concentrations of PSA-3 peptide (as compared with the 50 µg/ml used in the previous experiments described), uninfected DCs or DCs infected with FP-WT were unable to stimulate autologous T-cell IFN- production. At all concentrations of PSA peptide used, however, rF-TRICOM-infected DCs serving as APCs were capable of stimulating autologous T cells to produce IFN- (Fig. 5A)
. The ability of rF-TRICOM-infected DCs to activate T cells at IVS-4 to PSA was analyzed further by changing effector cell:APC ratios (Fig. 5B)
. Peptide-pulsed, rF-TRICOM-infected DCs serving as APCs were shown to be more effective in inducing IFN- production in autologous T cells as compared with uninfected or FP-WT control vector-infected DCs at all effector cell:DC ratios.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This is not the first study in which an APC has been modified to express a costimulatory molecule. Zajac et al. (49)
reported that MART-1 peptide-pulsed APCs infected with an inactivated rV virus encoding the CD80 or CD86 costimulatory molecule were able to generate tumor-specific CTLs more effectively than noninfected APCs. However, the studies reported here differ from the earlier study in the type of vector and the number of costimulatory molecules used, which leads one to question whether all three costimulatory molecules are actually necessary for the hyperstimulation of T cells observed. CD80, ICAM-1, and LFA-3 were chosen because of their different ligands on T cells and their unique ways of activating T cells (50, 51, 52)
. The peptide specificity of CTL activity precludes the effects of natural killer cells. Numerous studies have demonstrated that CD28 plays a key role in the delivery of signal 2 subsequent to engagement of either CD80 or CD86 (30
, 53, 54, 55)
. The LFA-3/CD2 pathway has been shown to initiate strong antigen-independent cell adhesion, substantially expand naïve T-helper cells, and induce IFN- production in memory cells. In turn, IFN- secretion can up-regulate the expression of ICAM-1 and CD80 molecules on APCs and allow for multiple adhesion pathways to amplify the immune response. The LFA-1/ICAM-1 pathway has been shown to stimulate adhesion and cell proliferation more efficiently in memory T-helper cells than in naïve cells. AP-1 and NF
B transcription factors are involved in the induction of several cytokine gene promoters and play a central role in the regulation of IL-2 gene transcription. CD80 costimulation can induce a large amount of NFB and AP-1 activity in T-helper cells, whereas LFA-3 costimulation can only moderately enhance AP-1 DNA binding activity and does not influence NFB activity induced by T-cell receptor engagement (1)
. Nonetheless, Ab-blocking experiments in the studies reported here were performed to determine whether all three costimulatory molecules were necessary for the activity observed. As shown in Table 5
, the use of anti-CD80, anti-CD54, or anti-CD58 alone decreased IFN- production in T cells stimulated with peptide-pulsed, TRICOM-infected DCs. The use of any combination of two of these antibodies further diminished IFN- production, and the use of all three antibodies completely eliminated IFN- production. These studies are also in agreement with studies using murine APCs and rV vectors expressing one, two, or three of the murine counterparts of CD80, ICAM-1, and LFA-3. For example, the use of rV-B7/ICAM was more effective in activating T cells than either rV-B7 or rV-ICAM, but it was not as effective as rV-TRICOM.
Previous studies have shown that when murine DCs are pulsed with OVA peptide and infected with either rV-TRICOM (mu) or rF-TRICOM (mu), they can enhance OVA-specific T-cell activation both in vitro and in vivo more effectively than peptide-pulsed DCs or peptide-pulsed, FP-WT-infected DCs (6
, 22)
. The studies reported here differ from the previous studies in several ways. First, because costimulatory molecules are species-specific, new vectors containing all three human costimulatory molecules were constructed and analyzed. As seen in Fig. 1
and Table 3
, all three molecules were efficiently expressed on the surface of human DCs several hours after infection. Major differences also exist in murine and human DCs. When murine bone-marrow precursor cells were treated with GM-CSF and IL-4, virtually all DCs expressed CD80, ICAM-1, and LFA-3. Infection of these murine DCs with TRICOM vectors was shown to enhance both the MFI of expression of each of the three costimulatory molecules and cell activation. In the studies reported here and in previous studies (9, 10, 11, 12, 13, 14, 15, 16, 17)
, human PBMCs treated with rhGM-CSF and rhIL-4 were shown to vary from patient to patient in their expression levels of these human costimulatory molecules. Moreover, the percentage of cells expressing costimulatory molecules are never as high as those observed with murine cells. Consequently, in the studies reported here, the use of the human TRICOM vector increased both the percentage of cells expressing all three costimulatory molecules as well as the MFI of expression levels of each of these molecules. As seen in Tables 2
and 3
, the additional maturation of DCs (which were treated previously with rhGM-CSF and rhIL-4) with rhTNF- or CD40L increased some of the classical human DC markers. In each case, however, infection of these DCs with TRICOM further enhanced the ability of the DCs to enhance T-cell activation. These studies are in agreement with studies reported previously that used murine DCs. In those studies (6
, 22)
, maturation of murine DCs with TNF-, CD40L-specific monoclonal Ab, or LPS slightly enhanced peptide-specific T-cell activation. Infection of these more-mature DCs with rF-TRICOM (mu) or rV-TRICOM (mu), however, markedly enhanced T-cell activation. As in the studies reported here, the use of a wild-type vector (FP-WT) alone had no effect on T-cell activation.
One of the concerns in infecting peptide-pulsed DCs with rF-TRICOM to hyperstimulate T cells is that too much stimulation will lead to T-cell apoptosis (56
, 57)
. As shown in Fig. 3
, this is not the case. Apoptosis levels in T cells stimulated with peptide-pulsed, rF-TRICOM-infected DCs were similar to levels in T cells stimulated with peptide-pulsed DCs. This may be explained by the fact that programmed cell death can be prevented by CD28-mediated costimulation, growth factors such as IL-2, and activation of survival-associated genes such as bcl-2 and bcl-Xl (58
, 59)
.
IL-12 is a cytokine produced by several types of APCs, including DCs. IL-12 is a major Th1-promoting factor and is essential in generating Th1 cells from naïve precursors. Moreover, IL-12 plays an important role in providing costimulatory and antiapoptotic signals that regulate the activity of effector-memory T cells (60) . Therefore, we asked if the infection of DCs with a fowlpox vector (either FP-WT or rF-TRICOM) would diminish IL-12 production. The production of IL-12 did not decrease in rF-TRICOM-infected DCs treated with LPS when compared with IL-12 production by uninfected DCs or vector control-infected DCs. A relatively high concentration of LPS (1 µg/ml) was used in these experiments because the DCs were cultured in serum-free medium (61 , 62) .
It is generally believed that DCs are the most potent of all APCs. However, this does not mean that strategies to enhance their potency cannot be devised. One can theorize that hundreds of thousands of years of evolution have placed the capacity of DCs to activate T cells into a "median state" between (a) their ability to activate T cells to specific microbiological pathogens and (b) their ability to induce autoimmunity to self-antigens. Thus, we theorize that the natural expression levels of several costimulatory molecules on human DCs of different states of maturity would accommodate this median state of efficacy in activating T cells. Because the fowlpox vector used here does not integrate and will express transgenes in the cytoplasm for only 2–3 weeks, at which time infected cells will die, one can readily control when and how to use rF-TRICOM DCs that are pulsed with a specific peptide, thus minimizing the risk of autoimmunity. However, one can never rule out that possibility.
Several practical applications can be derived from the studies reported here. One of these is the identification of new T-cell epitopes either of known antigens associated with infectious disease agents or tumors or of T-cell epitopes of newly discovered gene products. The studies reported here demonstrated that, in some cases, after only 24 h of incubation with PBMCs derived from a host, T cells can be activated with rF-TRICOM-infected DCs pulsed with a given peptide; the use of the same peptide-pulsed DCs without TRICOM infection did not generate a T-cell response. One can also potentially use rF-TRICOM-infected DCs to monitor responses to a given vaccine regimen. Finally, these studies indicate the feasibility of using peptide-pulsed, rF-TRICOM-infected DCs as a vaccine to prevent or treat infectious diseases, such as AIDS and malaria, or in immunotherapy protocols for preneoplastic or neoplastic conditions.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, MD 20892-1750. Phone: (301)496-4343; Fax: (301)496-2756; E-mail: js141c{at}nih.gov 
2 The abbreviations used are: APC, antigen-presenting cell; DC, dendritic cell; ICAM-1, intercellular adhesion molecule-1; LFA-3, leukocyte function-associated antigen-3; GM-CSF, granulocyte macrophage-colony stimulating factor; IL-4, interleukin-4; PBMC, peripheral blood mononuclear cell; TNF-, tumor necrosis factor-; TRICOM, triad of costimulatory molecules; OVA, ovalbumin; rF, recombinant fowlpox; rV, recombinant vaccinia; TAA, tumor-associated antigen; CEA, carcinoembryonic antigen; Flu, influenza; HPV, human papillomavirus; PSA, prostate-specific antigen; FP-WT, wild-type fowlpox virus; CM, complete medium; rh, recombinant human; MOI, multiplicity of infection; IVS, in vitro stimulation; TUNEL, terminal deoxynucleotidyl transferase-mediated nick-end labeling; MFI, mean fluorescent intensity; LPS, lipopolysaccharide; Ab, antibody; NFB, nuclear factor B.
Received 12/12/00. Accepted 2/28/01.
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), infected with rF-TRICOM (
), or infected with FP-WT as a control vector (
). The effector cell:APC ratio was 10:1. The 24-h culture supernatants were collected and screened for the secretion of IFN-
); DCs treated with rhGM-CSF and rhIL-4 for 7 days and then infected with FP-WT (
