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Immunology |
Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [J. W. H., H. S., M. G. O. L., J. S.], and Therion Biologics Corporation, Cambridge, Massachusetts 02142 [A. G. Y., L. G.]
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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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2 integrin) complex, and LFA-3 interacts with the CD2 (LFA-2) molecules. These molecules have been individually shown to costimulate T-cell proliferation in vitro (4)
. However, because they may be expressed simultaneously on APCs, it has been difficult to examine relative potencies of individual costimulatory molecules during the induction of T-cell proliferation (2)
. Because it has been proposed that both antigen and costimulatory molecules must be expressed in the same cell to properly engage the TCR and costimulatory receptor, respectively, an admixture of several recombinant vectors could be used to explore the potential cooperation of costimulatory molecules (6) . The disadvantage of this approach, however, is that the admixture of three or more vectors or viruses has a statistically diminished probability of coinfecting the same cell. Thus, a multigene construct would be preferable for expression of multiple costimulatory molecule genes in the same cell. The use of retroviral vectors, however, requires multiple drug selection procedures. We report here, for the first time, the development of constructs containing and expressing a TRICOM (B7-1, ICAM-1, and LFA-3). The synergistic effect of these costimulatory molecules on the enhanced activation of T cells is demonstrated. More specifically, these three costimulatory molecule genes have been inserted into two vectors: vaccinia, which is replication competent; and avipox (fowlpox), which is replication defective. In each case, the degree of T-cell activation using vectors containing three costimulatory molecules was far greater than the sum of the constructs, each containing one costimulatory molecule.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of Recombinant Viruses: Fluorescent Analysis of Protein Surface Expression.
Confluent MC38 cells, which are positive for class I (21)
, were infected with vaccinia constructs (V-WT, rV-B7-1, rV-ICAM-1, rV-LFA-3, and rV-TRICOM) or fowlpox constructs (WT-FP, rF-B7-1, rF-ICAM-1, and rF-CEA/TRICOM) at 5 MOI (pfu/cell) for 5 h. CEA was used in one rF construct as a marker gene only. After infection, cells were harvested and immunostained with FITC-conjugated MAbs specific for murine B7-1, ICAM-1, or CD48 (PharMingen, San Diego, CA). Cell fluorescence was analyzed with a FACScan cytometer (Becton Dickinson, Mountain View, CA) with the CellQuest program.
In Vitro Costimulation Analysis.
Female C57BL/6 mice (6–8 weeks of age) were obtained from Taconic Farms (Germantown, NY). Naive T cells were isolated as described previously (22)
. For certain experiments, T cells were further fractionated into CD4+ and CD8+ populations by negative selection using anti-CD4 or anti-CD8 paramagnetic beads (MiniMACS; Miltenyi Biotec, Auburn, CA). T cells were added at 105/well in 96-well, flat-bottomed plates (Costar, Cambridge, MA). Stimulator cells consisted of uninfected MC38 cells or cells infected for 5 h with 5 MOI of vaccinia constructs (V-WT, rV-B7-1, rV-ICAM-1, rV-LFA-3, and rV-TRICOM) or fowlpox constructs (WT-FP, rF-B7-1, rF-ICAM-1, and rF-CEA/TRICOM) fixed with 2% paraformaldehyde and added at 104/well. Cells in all wells were cultured in a total volume of 200 µl of complete media (22)
in the presence of several dilutions (5 to 0.625 µg/ml) of Con A (Sigma) for 2 days. Control wells received T cells, stimulator cells, and media only. For indicated experiments, plate-bound anti-CD3 (1.5 to 0.012 µg/well) was substituted for Con A. Cells were labeled for the final 12–18 h of the incubation with 1 µCi/well [3H]thymidine (New England Nuclear, Wilmington, DE) and harvested with a Tomtec cell harvester (Wallac Inc., Gaithersburg, MD). The incorporated radioactivity was measured by liquid scintillation counting (Wallac 1205 Betaplate; Wallac, Inc.) The results from triplicate wells were averaged and are reported as mean cpm ± SE. For indicated experiments, the in vitro costimulation analysis was performed in the presence of either a MAb specific for the expressed costimulatory molecule or the matching isotype control antibody (Armenian hamster IgG, polyclonal). Antibodies used to block T-cell proliferation were hamster anti-B7-1, hamster anti-ICAM, or hamster anti-CD48, all from PharMingen. All antibodies were used at 25 µg/ml final concentration.
C57BL/6 splenocytes were harvested and depleted of T cells by CD90 magnetic beads (Miltenyi Biotec). Spleen stimulator cell populations were prepared by infection with 25 MOI V-WT, rV-B7-1, or rV-TRICOM for 18 h, followed by irradiation (20 Gy). Allogeneic (BALB/c) or syngeneic (C57BL/6) responder T cells (105/well) were prepared as described above and cocultured with graded numbers of spleen stimulator cells for 3 days and labeled for the final 12–18 h of the incubation with 1 µCi/well [3H]thymidine.
In other experiments, OVA (Ovalbumin257–264, SIINFEKL)-specific responder T cells (105/well) were cocultured with MC38 stimulator cell populations (MC38 or MC38 infected with V-WT, rV-B7-1, or rV-TRICOM), prepared as described above, and irradiated (300 Gy). OVA-specific T cells (105/well) were cocultured with MC38 stimulator cells (104/well) in the presence of either OVA peptide or control peptide VSVN (vesicular stomatitis virus N52–59, RGYVYQGL) for 2 days and labeled for the final 12–18 h of the incubation with 1µCi/well [3H]thymidine. The incorporated radioactivity was measured by liquid scintillation counting.
Cytokine Analysis.
CD4+ and CD8+ T-cell populations were prepared as described above and added at 2.5 x 106/well in a six-well plate (Costar). Stimulator cell populations were prepared as above and added at 2.5 x 105/well. Cells were cultured in a total volume of 5 ml of complete media in the presence of 2.5 µg/ml Con A for 24 h. Supernatant fluids were collected and analyzed for murine IL-2, IFN-
, TNF-
, GM-CSF, and IL-4 by capture ELISA as described previously (23)
. Sensitivity of detection was 30, 100, 20, 20, and 20 pg/ml, respectively.
RNA populations from stimulated cells were also analyzed by multiprobe RNase protection assay. Defined riboprobes for murine cytokines were purchased from PharMingen. Assays were performed as described previously (24) . Radioactivity contained in bands on dried polyacrylamide gels was quantified using a Storm system PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The net cpm for a given band was calculated by the following formula [cpm of cytokine gene - cpm of background] and was expressed as a percentage of the housekeeping gene transcript L32.
Apoptosis Assay.
CD8+ cells were preincubated for 48 h in the presence of various stimulator cells as described in the in vitro costimulation analysis section and replated to 96-well plates for 24 h. Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, as described previously (25)
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In Vivo Studies.
Six- to eight-week-old female C57BL/6 mice (Taconic Farms) or C57BL/6 mice transgenic for human CEA (26)
were immunized by tail scarification with either HBSS or with 107 pfu of either rV-CEA, rV-CEA/B7-1, or rV-CEA/TRICOM. Lymphoproliferation activity of splenocytes was analyzed as described previously (22)
. In other experiments, C57BL/6 mice were immunized as above, challenged 100 days later with 106 MC38 cells expressing CEA (22)
, and monitored for survival.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Uninfected cells (data not shown) and cells infected with wild-type vaccinia virus (V-WT) failed to express any of the three costimulatory molecules (Fig. 1)
. This observation was confirmed by PCR (data not shown). In contrast, cells infected with rV-B7-1, rV-ICAM-1, or rV-LFA-3 became positive for their respective transgenes (Fig. 1)
. Similar analysis of a construct containing two costimulatory molecules (rV-B7-1/ICAM-1) showed expression of B7-1 (78% positive with a mean fluorescent intensity of 1012) and ICAM-1 (70% positive with a mean fluorescence intensity of 690). Moreover, cells infected with the vaccinia multiple-gene construct rV-TRICOM coexpressed all three costimulatory molecules (Fig. 1)
. Coexpression of all three costimulatory molecules on >79% of cells was confirmed by three-color cytometric analysis. To determine whether the recombinant fowlpox viruses expressed their recombinant proteins, MC38 cells were infected with the fowlpox constructs in a similar manner (Fig. 1)
. Again, cells infected with WT-FP failed to express any costimulatory molecule. Cells infected with rF-B7-1 became positive for B7-1 protein, and cells infected with rF-ICAM-1 became positive for ICAM-1 protein. A rF-LFA-3 vector was not constructed. However, cells infected with the fowlpox multiple-gene construct rF-CEA/TRICOM coexpressed all three costimulatory molecules (Fig. 1)
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or fowlpox (Fig. 2B)
viruses engineered to express costimulatory molecules. The second, or costimulatory, signal was delivered to the T cell via one or more costimulatory molecules expressed on the surface of these stimulator MC38 cells. As shown in Fig. 2A
, both uninfected MC38 cells and MC38/V-WT induced marginal proliferation of T cells at all levels of Con A examined. MC38/LFA-3 induced a small (2.1-fold) but significant (P < 0.05) increase in T-cell proliferation. Delivery of signal-2 via MC38/ICAM-1 induced a 3.5-fold increase in T-cell proliferation at 2.5 µg/ml Con A. MC38/B7-1 induced a 7.8- and 16-fold increase in proliferation at 2.5 and 1.25 µg/ml Con A, respectively. However, MC38/TRICOM (MC38 cells coexpressing all three costimulatory molecules) induced a 17.5-fold increase in T-cell proliferation at 2.5 µg/ml Con A and a 34-fold increase at 1.25 µg/ml Con A. Moreover, at low Con A levels (0.625 µg/ml), expression of ICAM-1 and LFA-3 did not induce T-cell proliferation. Although B7-1 induced measurable proliferation (20,000 cpm) at 0.625 µg/ml Con A, the coexpression of all three costimulatory molecules induced an even greater level of proliferation (100,000 cpm; Fig. 2A
). These experiments were repeated four times with similar results.
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. Again, uninfected MC38, MC38/WT-FP, or MC38/rF-CEA induced marginal proliferation of T cells at all levels of Con A examined. MC38/rF-ICAM-1 supported a 2-fold increase, MC38/rF-B7-1 supported a 3.2-fold increase, and MC38/rF-CEA/TRICOM supported a 6-fold increase in T-cell proliferation at 2.5 µg/ml Con A. Similar results were obtained when this experiment was repeated two additional times and when the first signal was delivered via immobilized anti-CD3 (data not shown). To further confirm the specificity of the proliferative contribution of B7-1, ICAM-1, or LFA-3, MC38 stimulator cells were prepared by infection with V-WT, rV-B7-1, rV-ICAM-1, or rV-LFA-3 and cocultured with naive murine T cells and Con A in the presence or absence of MAb specific for the given costimulatory molecule. MC38/B7-1 enhanced T-cell proliferation 4.5-fold more than that of MC38/V-WT. This increased proliferation was inhibited 83% by the addition of a blocking MAb for murine B7-1. Similarly, MC38/ICAM-1 increased proliferation 2.25-fold, which was then reduced by 88% in the presence of anti-murine ICAM-1 MAb. Finally, MC38/LFA-3 increased proliferation 2.1-fold, which was then reduced by 98% in the presence of antimurine CD48 MAb. For each group, incubation with the appropriate isotype control antibody failed to block the noted proliferation. This experiment was repeated two additional times with similar results.
Determination of Costimulatory Molecule Capacity.
Modification of the in vitro costimulation assay allowed a quantitative estimation of the relative capacity of B7-1, ICAM-1, and/or LFA-3 to deliver the second signal for T-cell proliferation. To that end, stimulator cells (MC38 cells infected with the various recombinant vaccinia viruses) were titered by dilution with varying amounts of MC38 cells infected with V-WT and cocultured with a constant number of T cells in the presence of 2.5 µg/ml Con A. The total MC38:T-cell ratio in these experiments remained constant at 1:10. As seen in Fig. 3
, MC38/LFA-3 enhanced proliferation of T cells over that of MC38/V-WT to a dilution of 40% (i.e., of the stimulator cells in the well, 40% were infected with rV-LFA-3 and the remaining 60% were infected with V-WT). MC38/ICAM-1 or MC38/B7-1 supported increased T-cell proliferation to dilutions of 13 and 6%, respectively. In contrast, MC38/TRICOM enhanced proliferation when <3% of stimulator cells contained the TRICOM vector (extrapolated to <1% via linear least squares analysis). Given the titration curves of these individual costimulatory molecules, it appeared that the extent of T-cell proliferation mediated by ICAM-1 and B7-1 is 3- and 6-fold, respectively, more potent than that mediated by LFA-3 alone. Clearly, the strongest proliferation, however, is mediated by TRICOM. It should be noted (Fig. 3)
that at relatively low stimulator cell concentrations (i.e., when 3–6% of the MC38 cells are acting as stimulator cells), expression of LFA-3, ICAM-1, and even B7-1 alone does not enhance T-cell activation, whereas the TRICOM-expressing stimulator cells substantially enhance T-cell activation. The data in Fig. 3
(inset) show proliferation results obtained when 3% of the MC38 stimulator cells were infected with the vectors denoted. Because each well contained 104 total MC38 cells and 105 naive T cells, the actual stimulator:T-cell ratio in these cultures was 0.003. Note that the MC38 cells infected with the two-gene construct (rV-B7-1/ICAM-1) induced little, if any, proliferation of T cells under these conditions, whereas MC38/TRICOM increased proliferation substantially (P < 0.0001).
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, respectively, show the proliferation of purified CD4+ and CD8+ cells activated with suboptimal concentrations of Con A. The stratification of stimulator cell effects on proliferation was similar for both CD4+ and CD8+ cells; MC38/LFA-3 stimulated the weakest proliferation, followed by MC38/ICAM-1 and MC38/B7-1. MC38/TRICOM were the most potent stimulator cells for both CD4+ and CD8+ T cells. These experiments were repeated three additional times with similar results. It should be noted that at very low concentrations of Con A (0.625 µg/ml; Fig. 4, C and D
), there was no significant enhancement in activation of CD4+ or CD8+ T cells when either ICAM-1, LFA-3, B7-1, or the B7-1/ICAM-1 dual recombinant was used to provide the second signal. However, substantial activation of both T-cell subsets was observed when the TRICOM vector was used. Similar results were noted when the first signal was delivered via immobilized anti-CD3 (data not shown).
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, C57BL/6 splenocytes infected with rV-TRICOM enhanced allospecific T-cell proliferation to far greater levels than uninfected splenocytes or splenocytes infected with V-WT or rV-B7-1. All stimulator cell populations, even those infected with TRICOM, that were incubated with syngeneic T cells resulted in no proliferation (<1000 cpm; Fig. 5A
).
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. Stimulator cells pulsed with control peptide VSVN did not support proliferation of the OVA-specific T-cell line (<1000 cpm).
Cytokine Studies.
It has been reported that B7-1 costimulation prolongs IL-2 mRNA half-life and up-regulation of IL-2 transcription, resulting in production of considerable amounts of secreted IL-2 (3
, 8) . Additionally, T-cell costimulation with LFA-3 has been reported to have an effect on a variety of cytokines, notably IL-2 and IFN- (4)
. To determine qualitative and quantitative effects of costimulation by single or multiple costimulatory molecules on cytokine production, purified CD4+ and CD8+ T cells were cocultured with various stimulator cells expressing either B7-1, ICAM-1 or LFA-3, or expressing all three molecules (TRICOM) in the presence of 2.5 µg/ml Con A. Supernatant fluids were analyzed for IL-2, IFN-, TNF-, GM-CSF, and IL-4 after 24 h (Fig. 6)
. Uninfected MC38 (data not shown) and MC38/V-WT induced a marginal quantity of IL-2 from CD4+ cells, whereas MC38/B7-1 induced 3979 pg/ml (Fig. 6A)
. However, T-cell stimulation with MC38/TRICOM induced a 10-fold greater amount of IL-2 (Fig. 6A)
. Similarly, MC38/B7-1 induced a marginal quantity of IL-2 from CD8+ cells, whereas MC38/TRICOM induced a 20-fold greater amount (6182 pg/ml; Fig. 6B
). IFN- production by stimulated T cells was also examined (Fig. 6, C and D)
. MC38/B7-1 and MC38/LFA-3 induced only moderate amounts of IFN- from CD4+ cells. In contrast, stimulation of CD4+ cells with MC38/TRICOM induced 4-fold more IFN- than stimulation with any other construct (Fig. 6C)
. Stimulation of CD8+ cells with MC38/TRICOM induced the greatest amount of IFN-, greater than 6-fold more than CD8+ cells stimulated with any of the other constructs (Fig. 6D)
. Stimulation of either cell type with any construct failed to mediate significant changes (P > 0.05) in the levels of secreted TNF-, GM-CSF, or IL-4 (data not shown). It appears that the predominant culmination of stimulation via the TRICOM construct was IL-2 secretion from CD4+ cells and IFN- secretion from CD8+ T cells. These experiments were repeated three additional times with similar results. Studies were also carried out comparing stimulator cells infected with the two-gene construct (rV-B7-1/ICAM-1) with the triad construct (rV-TRICOM) for their ability to enhance cytokine production by T cells. Only small differences were observed between the two in IFN- production by either CD4+ or CD8+ cells or in IL-2 production by CD8+ cells. However, a substantial difference was seen in the stimulation of IL-2 production by CD4+ cells (5000 pg/ml using MC38/B7-1/ICAM-1 versus 39600 pg/ml using MC38/TRICOM).
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. These experiments were repeated twice with similar results. Levels of IL-4, IL-5, IL-10, IL-15, and IL-6 were similar in CD4+ T cells stimulated with MC38/V-WT, MC38/B7-1, MC38/ICAM-1, MC38/LFA-3, or MC38/TRICOM (Fig. 7, A–B)
. IL-2 and IFN- expression levels were highest in CD4+ T cells stimulated with MC38/TRICOM when compared with CD4+ cells stimulated with MC38 cells expressing any single costimulatory molecule (Fig. 7, A and B)
. Slightly higher levels of IL-13, IL-9, and IL-6 were also noted in CD4+ cells stimulated with MC38/TRICOM. Expression of cytokine genes was also analyzed in stimulated CD8+ T cells. Of the cytokine RNAs analyzed, IL-2 and, in particular, IFN- levels were significantly higher when these cells were stimulated with MC38/TRICOM, compared with T cells stimulated with MC38 cells expressing any single costimulatory molecule (Fig. 7, A and C)
. Thus, the predominant synergistic effect of the triad of costimulatory molecules in cytokine production was IL-2 in CD4+ cells and IFN- in CD8+ T cells.
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). T cells activated by Con A and MC38/B7-1 or Con A and MC38/TRICOM exhibited substantially less spontaneous apoptosis (31.3 ± 3.8 and 30.7 ± 1, respectively; Fig. 8, C and D
).
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(inset), splenic T cells of mice vaccinated with rV-TRICOM showed higher levels of CEA-specific stimulation compared with T cells obtained from mice vaccinated with rV-CEA; ovalbumin and Con A were used as controls. An experiment was then conducted to determine whether rV-CEA/TRICOM could induce long-term immunity. Mice (five/group) were vaccinated one time with either V-WT, rV-CEA, or rV-CEA/TRICOM. One hundred days later, mice were challenged with a high dose (1 x 106) of MC38 colon carcinoma cells expressing CEA (22)
. All mice receiving V-WT and rV-CEA succumbed to tumors, whereas all mice vaccinated with rV-TRICOM were alive 50 days after challenge (Fig. 9)
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. Responses to ovalbumin and Con A were used as controls. The remaining three CEA-transgenic mice in each group were used to determine whether antitumor responses to a CEA-expressing tumor could be enhanced using a TRICOM vector. These mice were first inoculated s.c. with 4 x 105 MC38 carcinoma cells expressing the CEA gene (22)
. Four days later, mice were vaccinated one time at a distal site with 107 pfu viral recombinant or buffer. No tumors grew in mice vaccinated with rV-CEA/TRICOM, whereas tumors continued to grow in mice vaccinated with buffer, rV-CEA and rV-CEA/B7-1 (Table 1)
. Although preliminary, these results support the in vivo activity of TRICOM vectors.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several groups have investigated the cooperation of two of these molecules in T-cell costimulation. Dubey et al. (9)
have reported that costimulation by both B7-1 and ICAM-1 is a prerequisite for naive T-cell activation, whereas Cavallo et al. (10)
determined that B7-1 and ICAM-1 must by coexpressed by tumor cells to establish an antitumor memory response. In addition, costimulation by B7-1 and LFA-3 has been shown to act additively both upon T-cell proliferation and cytokine production (4
, 7)
. These previous studies were carried out using two costimulatory molecules and retroviral vectors. One gene was transduced into the target cell line and drug selected. These cells were then transduced again with a second recombinant retroviral construct, followed by selection with a different agent. This process often requires weeks or months. Using recombinant poxvirus vectors, one is able to achieve the coexpression of three costimulatory molecules 5 h after infection (Fig. 1)
. In vitro MC38 cells infected with either recombinant vaccinia or avipox TRICOM vectors were shown to enhance proliferation of T cells to a much greater extent than MC38 cells infected with vectors containing the gene for any single costimulatory molecule (Fig. 2)
. In addition, the relative strength of the second signal delivered to the T cell by the combination of costimulatory molecules appeared to be far greater than that delivered by MC38 cells expressing any single costimulatory molecule (Fig. 3)
. Dubey et al. (9)
have demonstrated that at low stimulator:T-cell ratios, synergy was noted with B7-1 and ICAM-1. Our studies confirm these findings. However, at extremely low stimulator:T-cell ratios (Fig. 3)
or weak signal 1 (0.625 µg/ml Con A; Fig. 4, C and D
), the two-gene construct (rV-B7-1/ICAM-1) had little effect, if any, on proliferation. In contrast, stimulation via the TRICOM construct had a substantial and statistically significant effect on proliferation. The predominant effect of stimulation via the TRICOM construct was IL-2 production from CD4+ cells and IFN- production from CD8+ T cells (Fig. 6)
, whereas few type 2 cytokines, if any, were produced. Cytokine expression analysis by RNase protection provided a profile compatible with the in vitro cytokine assay, manifested by substantially higher expression of IL-2 and IFN- in both CD4+ and CD8+ T cells stimulated with all three costimulatory molecules, compared with stimulation by any single costimulatory molecule (Fig. 7)
. These data are in accordance with previous studies demonstrating that in the context of low CD28 costimulation, T cells produced low levels of IL-1, whereas strong CD28 costimulation supported production of IL-2, IFN-, and IL-13 (28)
. Taken together, the studies reported here indicate that optimal naive T-cell responses require a higher level of costimulation than was previously thought, and that this could be provided by the combined action of three costimulatory molecules.
One question that is immediately raised is the potential of overstimulated T cells to undergo PCD. The results shown in Fig. 8, A and B
, clearly demonstrate apoptosis in T cells stimulated with MC38 cells in the presence of Con A with or without V-WT infection (i.e., in the absence of signal 2). Whereas Con A with MC38/TRICOM clearly stimulated CD8+ cells to far greater levels than Con A with MC38/B7-1 (Fig. 2)
and resulted in the production of higher levels of IFN- and IL-2 (Figs. 6
and 7
), this did not result in any greater degree of apoptosis (Fig. 8D)
. Our results are in agreement with those of previous studies, which found that costimulation through the CD28 receptor appears to play an important role in enhancing the resistance of activated T cells to undergo PCD in culture (29)
. This could be attributed to augmentation of cytokine production by these cells and potential up-regulation of survival genes. Further studies are presently under way to analyze the detailed mechanism of survival in these cells.
Perhaps the most studied T-cell costimulatory molecule is B7-1. This molecule’s ability to enhance T-cell activation using retroviral vectors, anti-CTLA-4 antibodies, and poxvirus vectors is well established. The studies reported here rank the order of T-cell stimulation by a single costimulatory molecule as B7-1 > ICAM-1 > LFA-3. However, the use of three costimulatory molecules was far superior to B7-1 alone in both proliferation and cytokine production for both CD4+ and CD8+ T cells. The studies reported here demonstrate the power of the multicostimulatory molecule effect to enhance T-cell proliferation in three very different systems and to use both naive and effector T-cell populations as responder cells. These three model systems are: (a) the activation of naive T cells using Con A or anti-CD3 as signal 1 (Fig. 2)
; (b) the use of OVA peptide as signal 1 and the activation of OVA-specific "effector" T cells from an established cell line (Fig. 5B)
; and (c) enhanced allospecific reactivity in a mixed lymphocyte reaction (Fig. 5A)
.
Initial in vivo experiments reported here indicate that rV-CEA/TRICOM is more efficient than rV-CEA/B7-1 in the induction of CEA-specific T-cell responses in both intact C57BL/6 mice and in CEA-transgenic C57BL/6 mice. Induction of long-term immunity to tumor challenge is indicated for rV-CEA/TRICOM in C57BL/6 mice (Fig. 9)
, and antitumor activity for rV-CEA/TRICOM versus rV-CEA/B7-1 or rV-CEA in CEA transgenic mice is indicated in Table 1
. However, only limited amounts of the CEA transgenic mice were available and used in these studies. Additional comprehensive studies with more CEA transgenic mice and different doses and dose schedules of rV-TRICOM, rV-CEA/B7-1, and rV-CEA, along with different tumor burdens, are clearly indicated. These mice will also have to be carefully evaluated for evidence of autoimmunity. Because of the scarcity of CEA-transgenic mice, these will be long-term experiments.
There are several possible mechanisms for the synergy observed here between B7-1, ICAM-1, and LFA-3. The ICAM-1/LFA-1 interaction reportedly costimulates the TCR-mediated activation of T cells by sustaining the increase in the same intracellular second messengers as generated by TCR engagement. The ICAM-1/LFA-1 interaction is necessary to up-regulate expression of the IL-2R- chain and CD28 on T cells, which is required to render them competent to respond to IL-2 and B7-1 costimulation. The B7-1/CD28 interaction delivers a TCR-independent costimulatory signal that increases both transcriptionally and posttranscriptionally the expression of IL-2 and other immunoregulatory lymphokines. The LFA-3/CD2 interaction induces tyrosine phosphorylation of several intracellular second messengers, Ca2+ mobilization, and cAMP production, resulting in elaboration of a variety of cytokines, notably IL-2 and IFN- (4)
. Thus, it appears that the three costimulatory molecules could be cooperating by enhancing the antigen-dependent activation of T cells, as well as their production of and response to autocrine and paracrine growth factors. Future studies should involve an analysis of the signal transduction pathways induced by T-cell costimulation via stimulator cell populations expressing B7-1, ICAM-1, and LFA-3, alone or in combination.
In conclusion, these studies demonstrate for the first time the ability of vectors to introduce three costimulatory molecules into a cell and to rapidly and efficiently activate both CD4+ and CD8+ T-cell populations to levels far greater than those achieved when any one or two of these costimulatory molecules are used. This new threshold of T-cell activation, with the caveat of autoimmunity mentioned above, has broad implications in vaccine design and development for a range of diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, c/o Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, 10 Center Drive, Room 8B07, Bethesda, MD 20892. Phone: (301) 496-4343; Fax: (301) 496-2756; E-mail: js141c{at}nih.gov 
2 The abbreviations used are: APC, antigen-presenting cell; TCR, T-cell receptor; ICAM, intercellular adhesion molecule; LFA, leukocyte function-associated antigen; TRICOM, TRIad of COstimulatory Molecules; CEA, carcinoembryonic antigen; MOI, multiplicity of infection; pfu, plaque-forming unit(s); MAb, monoclonal antibody; IL, interleukin; TNF, tumor necrosis factor; GM-CSF, granulocyte/macrophage-colony stimulating factor; PCD, programmed cell death.
Received 7/ 8/99. Accepted 10/ 1/99.
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P. C.R. Emtage, A. S.Y. Lo, E. M. Gomes, D. L. Liu, R. M. Gonzalo-Daganzo, and R. P. Junghans Second-Generation Anti-Carcinoembryonic Antigen Designer T Cells Resist Activation-Induced Cell Death, Proliferate on Tumor Contact, Secrete Cytokines, and Exhibit Superior Antitumor Activity In vivo: A Preclinical Evaluation Clin. Cancer Res., December 15, 2008; 14(24): 8112 - 8122. [Abstract] [Full Text] [PDF]
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E. K. Wansley, M. Chakraborty, K. W. Hance, M. B. Bernstein, A. L. Boehm, Z. Guo, D. Quick, A. Franzusoff, J. W. Greiner, J. Schlom, et al. Vaccination with a Recombinant Saccharomyces cerevisiae Expressing a Tumor Antigen Breaks Immune Tolerance and Elicits Therapeutic Antitumor Responses Clin. Cancer Res., July 1, 2008; 14(13): 4316 - 4325. [Abstract] [Full Text] [PDF]
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C. T. Garnett, J. Schlom, and J. W. Hodge Combination of Docetaxel and Recombinant Vaccine Enhances T-Cell Responses and Antitumor Activity: Effects of Docetaxel on Immune Enhancement Clin. Cancer Res., June 1, 2008; 14(11): 3536 - 3544. [Abstract] [Full Text] [PDF]
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J. L. Gulley, P. M. Arlen, K.-Y. Tsang, J. Yokokawa, C. Palena, D. J. Poole, C. Remondo, V. Cereda, J. L. Jones, M. P. Pazdur, et al. Pilot Study of Vaccination with Recombinant CEA-MUC-1-TRICOM Poxviral-Based Vaccines in Patients with Metastatic Carcinoma Clin. Cancer Res., May 15, 2008; 14(10): 3060 - 3069. [Abstract] [Full Text] [PDF]
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 J. Schlom, J. L. Gulley, and P. M. Arlen Paradigm Shifts in Cancer Vaccine Therapy Experimental Biology and Medicine, May 1, 2008; 233(5): 522 - 534. [Abstract] [Full Text] [PDF]
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 S. Mostbock, M. E. C. Lutsiak, D. E. Milenic, K. Baidoo, J. Schlom, and H. Sabzevari IL-2/Anti-IL-2 Antibody Complex Enhances Vaccine-Mediated Antigen-Specific CD8+ T Cell Responses and Increases the Ratio of Effector/Memory CD8+ T Cells to Regulatory T Cells J. Immunol., April 1, 2008; 180(7): 5118 - 5129. [Abstract] [Full Text] [PDF]
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 L. Tian, J. Lappalainen, M. Autero, S. Hanninen, H. Rauvala, and C. G. Gahmberg Shedded neuronal ICAM-5 suppresses T-cell activation Blood, April 1, 2008; 111(7): 3615 - 3625. [Abstract] [Full Text] [PDF]
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 J. Schlom, P. M. Arlen, and J. L. Gulley Immunotherapies with Other Therapeutic Modalities: New Paradigms for Clinical Trial Design ASCO Educational Book, January 1, 2008; 2008(1): 101 - 106. [Abstract] [Full Text] [PDF]
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M. T. Litzinger, R. Fernando, T. J. Curiel, D. W. Grosenbach, J. Schlom, and C. Palena IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity Blood, November 1, 2007; 110(9): 3192 - 3201. [Abstract] [Full Text] [PDF]
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S. Mostbock, S. Vidal, J. Schlom, and H. Sabzevari Enhanced Levels of Costimulation Lead to Reduced Effector/Memory CD8+ T Cell Functionality J. Immunol., September 15, 2007; 179(6): 3524 - 3534. [Abstract] [Full Text] [PDF]
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J. Schlom, P. M. Arlen, and J. L. Gulley Cancer Vaccines: Moving Beyond Current Paradigms Clin. Cancer Res., July 1, 2007; 13(13): 3776 - 3782. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, E. K. Wansley, M. E. Gruys, R. Wiltrout, J. Schlom, and J. W. Hodge Combination Therapy of an Orthotopic Renal Cell Carcinoma Model Using Intratumoral Vector-Mediated Costimulation and Systemic Interleukin-2 Clin. Cancer Res., March 15, 2007; 13(6): 1936 - 1946. [Abstract] [Full Text] [PDF]
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 C. P. Tarassoff, P. M. Arlen, and J. L. Gulley Therapeutic vaccines for prostate cancer. Oncologist, May 1, 2006; 11(5): 451 - 462. [Abstract] [Full Text] [PDF]
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P. M. Arlen, J. L. Gulley, C. Parker, L. Skarupa, M. Pazdur, D. Panicali, P. Beetham, K. Y. Tsang, D. W. Grosenbach, J. Feldman, et al. A Randomized Phase II Study of Concurrent Docetaxel Plus Vaccine Versus Vaccine Alone in Metastatic Androgen-Independent Prostate Cancer Clin. Cancer Res., February 15, 2006; 12(4): 1260 - 1269. [Abstract] [Full Text] [PDF]
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C. Palena, K. A. Foon, D. Panicali, A. G. Yafal, J. Chinsangaram, J. W. Hodge, J. Schlom, and K. Y. Tsang Potential approach to immunotherapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity of CLL cells via infection with vectors encoding for multiple costimulatory molecules Blood, November 15, 2005; 106(10): 3515 - 3523. [Abstract] [Full Text] [PDF]
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 R. D. Loberg, C. J. Logothetis, E. T. Keller, and K. J. Pienta Pathogenesis and Treatment of Prostate Cancer Bone Metastases: Targeting the Lethal Phenotype J. Clin. Oncol., November 10, 2005; 23(32): 8232 - 8241. [Abstract] [Full Text] [PDF]
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H. L. Kaufman and C. R. Divgi Optimizing Prostate Cancer Treatment by Combining Local Radiation Therapy with Systemic Vaccination Clin. Cancer Res., October 1, 2005; 11(19): 6757 - 6762. [Full Text] [PDF]
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S. Yang, J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL J. Immunol., September 15, 2005; 175(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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 K. J. Pienta and D. C. Smith Advances in Prostate Cancer Chemotherapy: A New Era Begins CA Cancer J Clin, September 1, 2005; 55(5): 300 - 318. [Abstract] [Full Text] [PDF]
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S. Yang, K.-Y. Tsang, and J. Schlom Induction of Higher-Avidity Human CTLs by Vector-Mediated Enhanced Costimulation of Antigen-Presenting Cells Clin. Cancer Res., August 1, 2005; 11(15): 5603 - 5615. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, K. Camphausen, C. N. Coleman, and J. W. Hodge The Requirement of Multimodal Therapy (Vaccine, Local Tumor Radiation, and Reduction of Suppressor Cells) to Eliminate Established Tumors Clin. Cancer Res., June 15, 2005; 11(12): 4533 - 4544. [Abstract] [Full Text] [PDF]
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P. L. Triozzi, K. O. Allen, R. R. Carlisle, M. Craig, A. F. LoBuglio, and R. M. Conry Phase I Study of the Intratumoral Administration of Recombinant Canarypox Viruses Expressing B7.1 and Interleukin 12 in Patients with Metastatic Melanoma Clin. Cancer Res., June 1, 2005; 11(11): 4168 - 4175. [Abstract] [Full Text] [PDF]
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J. W. Hodge, M. Chakraborty, C. Kudo-Saito, C. T. Garnett, and J. Schlom Multiple Costimulatory Modalities Enhance CTL Avidity J. Immunol., May 15, 2005; 174(10): 5994 - 6004. [Abstract] [Full Text] [PDF]
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M. A. Morse, T. M. Clay, A. C. Hobeika, T. Osada, S. Khan, S. Chui, D. Niedzwiecki, D. Panicali, J. Schlom, and H. K. Lyerly Phase I Study of Immunization with Dendritic Cells Modified with Fowlpox Encoding Carcinoembryonic Antigen and Costimulatory Molecules Clin. Cancer Res., April 15, 2005; 11(8): 3017 - 3024. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, and J. W. Hodge Induction of an Antigen Cascade by Diversified Subcutaneous/Intratumoral Vaccination Is Associated with Antitumor Responses Clin. Cancer Res., March 15, 2005; 11(6): 2416 - 2426. [Abstract] [Full Text] [PDF]
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K. Y. Tsang, C. Palena, J. Yokokawa, P. M. Arlen, J. L. Gulley, G. P. Mazzara, L. Gritz, A. Gomez Yafal, S. Ogueta, P. Greenhalgh, et al. Analyses of Recombinant Vaccinia and Fowlpox Vaccine Vectors Expressing Transgenes for Two Human Tumor Antigens and Three Human Costimulatory Molecules Clin. Cancer Res., February 15, 2005; 11(4): 1597 - 1607. [Abstract] [Full Text] [PDF]
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J. L. Marshall, J. L. Gulley, P. M. Arlen, P. K. Beetham, K.-Y. Tsang, R. Slack, J. W. Hodge, S. Doren, D. W. Grosenbach, J. Hwang, et al. Phase I Study of Sequential Vaccinations With Fowlpox-CEA(6D)-TRICOM Alone and Sequentially With Vaccinia-CEA(6D)-TRICOM, With and Without Granulocyte-Macrophage Colony-Stimulating Factor, in Patients With Carcinoembryonic Antigen-Expressing Carcinomas J. Clin. Oncol., February 1, 2005; 23(4): 720 - 731. [Abstract] [Full Text] [PDF]
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 Q. Li, A. L. Carr, E. J. Donald, J. J. Skitzki, R. Okuyama, L. M. Stoolman, and A. E. Chang Synergistic Effects of IL-12 and IL-18 in Skewing Tumor-Reactive T-Cell Responses Towards a Type 1 Pattern Cancer Res., February 1, 2005; 65(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
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H. K. Lyerly Your Ad here: optimizing adenoviral vector-based vaccines Blood, November 1, 2004; 104(9): 2612 - 2613. [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, C. N. Coleman, K. Camphausen, J. Schlom, and J. W. Hodge External Beam Radiation of Tumors Alters Phenotype of Tumor Cells to Render Them Susceptible to Vaccine-Mediated T-Cell Killing Cancer Res., June 15, 2004; 64(12): 4328 - 4337. [Abstract] [Full Text] [PDF]
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H. E. Zeytin, A. C. Patel, C. J. Rogers, D. Canter, S. D. Hursting, J. Schlom, and J. W. Greiner Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice Cancer Res., May 15, 2004; 64(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
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C. Kudo-Saito, J. Schlom, and J. W. Hodge Intratumoral Vaccination and Diversified Subcutaneous/ Intratumoral Vaccination with Recombinant Poxviruses Encoding a Tumor Antigen and Multiple Costimulatory Molecules Clin. Cancer Res., February 1, 2004; 10(3): 1090 - 1099. [Abstract] [Full Text] [PDF]
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J. W. Hodge, D. J. Poole, W. M. Aarts, A. Gomez Yafal, L. Gritz, and J. Schlom Modified Vaccinia Virus Ankara Recombinants Are as Potent as Vaccinia Recombinants in Diversified Prime and Boost Vaccine Regimens to Elicit Therapeutic Antitumor Responses Cancer Res., November 15, 2003; 63(22): 7942 - 7949. [Abstract] [Full Text] [PDF]
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M. Chakraborty, S. I. Abrams, K. Camphausen, K. Liu, T. Scott, C. N. Coleman, and J. W. Hodge Irradiation of Tumor Cells Up-Regulates Fas and Enhances CTL Lytic Activity and CTL Adoptive Immunotherapy J. Immunol., June 15, 2003; 170(12): 6338 - 6347. [Abstract] [Full Text] [PDF]
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J. W. Hodge, D. W. Grosenbach, W. M. Aarts, D. J. Poole, and J. Schlom Vaccine Therapy of Established Tumors in the Absence of Autoimmunity Clin. Cancer Res., May 1, 2003; 9(5): 1837 - 1849. [Abstract] [Full Text] [PDF]
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 J. Briones, J. M. Timmerman, D. L. Panicalli, and R. Levy Antitumor Immunity After Vaccination With B Lymphoma Cells Overexpressing a Triad of Costimulatory Molecules J Natl Cancer Inst, April 2, 2003; 95(7): 548 - 555. [Abstract] [Full Text] [PDF]
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J. W. Greiner, H. Zeytin, M. R. Anver, and J. Schlom Vaccine-based Therapy Directed against Carcinoembryonic Antigen Demonstrates Antitumor Activity on Spontaneous Intestinal Tumors in the Absence of Autoimmunity Cancer Res., December 1, 2002; 62(23): 6944 - 6951. [Abstract] [Full Text] [PDF]
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W. M. Aarts, J. Schlom, and J. W. Hodge Vector-based Vaccine/Cytokine Combination Therapy to Enhance Induction of Immune Responses to a Self-Antigen and Antitumor Activity Cancer Res., October 15, 2002; 62(20): 5770 - 5777. [Abstract] [Full Text] [PDF]
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E. S. Kass, J. W. Greiner, J. A. Kantor, K. Y. Tsang, F. Guadagni, Z. Chen, B. Clark, R. D. Pascalis, J. Schlom, and C. Van Waes Carcinoembryonic Antigen as a Target for Specific Antitumor Immunotherapy of Head and Neck Cancer Cancer Res., September 1, 2002; 62(17): 5049 - 5057. [Abstract] [Full Text] [PDF]
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 Y. Hu, J. Lee, J. A. McCart, H. Xu, B. Moss, H. R. Alexander, and D. L. Bartlett Yaba-Like Disease Virus: an Alternative Replicating Poxvirus Vector for Cancer Gene Therapy J. Virol., November 1, 2001; 75(21): 10300 - 10308. [Abstract] [Full Text] [PDF]
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G. Zheng, A. Chen, R. E. Sterner, P. J. Zhang, T. Pan, N. Kiyatkin, and M. L. Tykocinski Induction of Antitumor Immunity via Intratumoral Tetra-Costimulator Protein Transfer Cancer Res., November 1, 2001; 61(22): 8127 - 8134. [Abstract] [Full Text] [PDF]
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R. Xiang, F. J. Primus, J. M. Ruehlmann, A. G. Niethammer, S. Silletti, H. N. Lode, C. S. Dolman, S. D. Gillies, and R. A. Reisfeld A Dual-Function DNA Vaccine Encoding Carcinoembryonic Antigen and CD40 Ligand Trimer Induces T Cell-Mediated Protective Immunity Against Colon Cancer in Carcinoembryonic Antigen-Transgenic Mice J. Immunol., October 15, 2001; 167(8): 4560 - 4565. [Abstract] [Full Text] [PDF]
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K. Y. Tsang, M. Zhu, J. Even, J. Gulley, P. Arlen, and J. Schlom The Infection of Human Dendritic Cells with Recombinant Avipox Vectors Expressing a Costimulatory Molecule Transgene (CD80) to Enhance the Activation of Antigen-specific Cytolytic T Cells Cancer Res., October 1, 2001; 61(20): 7568 - 7576. [Abstract] [Full Text] [PDF]
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 R. T. Semnani, H. Sabzevari, R. Iyer, and T. B. Nutman Filarial Antigens Impair the Function of Human Dendritic Cells during Differentiation Infect. Immun., September 1, 2001; 69(9): 5813 - 5822. [Abstract] [Full Text] [PDF]
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D. W. Grosenbach, J. C. Barrientos, J. Schlom, and J. W. Hodge Synergy of Vaccine Strategies to Amplify Antigen-specific Immune Responses and Antitumor Effects Cancer Res., June 1, 2001; 61(11): 4497 - 4505. [Abstract] [Full Text] [PDF]
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M. Zhu, H. Terasawa, J. Gulley, D. Panicali, P. Arlen, J. Schlom, and K. Y. Tsang Enhanced Activation of Human T Cells via Avipox Vector-mediated Hyperexpression of a Triad of Costimulatory Molecules in Human Dendritic Cells Cancer Res., May 1, 2001; 61(9): 3725 - 3734. [Abstract] [Full Text]
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M. von Mehren, P. Arlen, J. Gulley, A. Rogatko, H. S. Cooper, N. J. Meropol, R. K. Alpaugh, M. Davey, S. McLaughlin, M. T. Beard, et al. The Influence of Granulocyte Macrophage Colony-Stimulating Factor and Prior Chemotherapy on the Immunological Response to a Vaccine (ALVAC-CEA B7.1) in Patients with Metastatic Carcinoma Clin. Cancer Res., May 1, 2001; 7(5): 1181 - 1191. [Abstract] [Full Text]
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H. Sabzevari, J. Kantor, A. Jaigirdar, Y. Tagaya, M. Naramura, J. W. Hodge, J. Bernon, and J. Schlom Acquisition of CD80 (B7-1) by T Cells J. Immunol., February 15, 2001; 166(4): 2505 - 2513. [Abstract] [Full Text] [PDF]
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E. Kass, D. L. Panicali, G. Mazzara, J. Schlom, and J. W. Greiner Granulocyte/Macrophage-Colony Stimulating Factor Produced by Recombinant Avian Poxviruses Enriches the Regional Lymph Nodes with Antigen-presenting Cells and Acts as an Immunoadjuvant Cancer Res., January 1, 2001; 61(1): 206 - 214. [Abstract] [Full Text]
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J. L. Marshall, R. J. Hoyer, M. A. Toomey, K. Faraguna, P. Chang, E. Richmond, J. E. Pedicano, E. Gehan, R. A. Peck, P. Arlen, et al. Phase I Study in Advanced Cancer Patients of a Diversified Prime-and-Boost Vaccination Protocol Using Recombinant Vaccinia Virus and Recombinant Nonreplicating Avipox Virus to Elicit Anti-Carcinoembryonic Antigen Immune Responses J. Clin. Oncol., December 1, 2000; 18(23): 3964 - 3973. [Abstract] [Full Text] [PDF]
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J. W. Hodge, A. N. Rad, D. W. Grosenbach, H. Sabzevari, A. G. Yafal, L. Gritz, and J. Schlom Enhanced Activation of T Cells by Dendritic Cells Engineered to Hyperexpress a Triad of Costimulatory Molecules J Natl Cancer Inst, August 2, 2000; 92(15): 1228 - 1239. [Abstract] [Full Text] [PDF]
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), rV-LFA-3 (
), rV-ICAM-1 or rF-ICAM-1 (•), rV-B7-1 or rF-B7-1 (
), and rV-TRICOM or rF-CEA/TRICOM (
). 
, uninfected MC38 cells. Bars, SD.
). B, OVA257–264-specific CD8+ T cells were cocultured with uninfected MC38 cells (not shown) or cells infected with V-WT (
