A Triad of Costimulatory Molecules Synergize to Amplify T‐‐Cell Activation in Both Vector‐‐Based and Vector‐‐Infected Dendritic Cell Vaccines

Figures & data

Figure 1. Effect of multiple costimulatory molecules on T cell proliferation. Naive murine T cells, in the presence of varying concentrations of Con A to provide the first signal, were co‐‐cultured with MC38 stimulator cells infected with either recombinant vaccinia ((A)) or recombinant fowlpox ((B)) vectors. Recombinant vectors were wild‐‐type ((i.e., V‐‐WT or WT‐‐FP [[open squares]])), rV‐‐LFA‐‐3 ((closed triangle)), rV‐‐ICAM‐‐1 or rF‐‐ICAM‐‐1 ((closed circles)), rV‐‐B7‐‐1 or rF‐‐B7‐‐1 ((closed diamonds)), and rV‐‐TRICOM or rF‐‐CEA//TRICOM ((closed squares)). Uninfected MC38 cells are open circles. Bars, SD. These data are as presented in Hodge et al. (([1999])).

Figure 2. Effect of costimulation on specific T‐‐cell populations. Murine CD4⁺⁺ ((A)) or CD8⁺⁺ T cells ((B)) were co‐‐cultured with uninfected MC38 cells ((open circle)), or cells infected with V‐‐WT ((open squares)), rV‐‐LFA‐‐3 ((closed triangles)), rV‐‐ICAM‐‐1 ((closed circles)), rV‐‐B7‐‐1 ((closed diamonds)) or rV‐‐TRICOM ((closed squares)) at a 10:1 ratio for 48 h in the presence of various concentrations of Con A. C and D show the proliferative responses of purified CD4⁺⁺ and CD8⁺⁺ cells, respectively, when co‐‐cultured in the presence of vector‐‐infected MC38 stimulator cells at a low Con A concentration ((0.625 μg//ml)). Bars, SD. These data are as presented in Hodge et al. (([1999])).

Figure 3. Effect of costimulation on cytokine RNA expression. A, murine CD4⁺⁺ or CD8⁺⁺ T cells were co‐‐cultured with MC38 stimulator cells infected with V‐‐WT ((lane A)), rV‐‐B7‐‐1 ((lane B)), rV‐‐ICAM‐‐1 ((lane C)), rV‐‐LFA‐‐3 ((lane D)), or rV‐‐TRICOM ((lane E)) at a T‐‐cell:stimulator‐‐cell ratio of 10:1 for 24 h in the presence of 2.5 μg//ml Con A. After culture, T‐‐cell RNA was analyzed by multiprobe Rnase protection assay. The quantitative representation of results from the autoradiograph is normalized for expression of the housekeeping gene L32 in B ((CD4⁺⁺ cells)) and C ((CD8⁺⁺ cells)). Order of histogram columns ((from left to right)): MC38//V‐‐WT, MC38//B7‐‐1, MC38//ICAM‐‐1, MC38//LFA‐‐3, and MC38//TRICOM. These data are as presented in Hodge et al. (([1999])).

Figure 4. Apoptosis of CD8⁺⁺ cells activated by Con A and either MC38 ((A)), MC38//V‐‐WT ((B)), MC38//B7‐‐1 ((C)), or MC38//TRICOM ((D)). Each panel depicts the level of apoptotic cells ((above line)) in each group as measured by the terminal deoxynucleotidyl transferase‐‐mediated nick end labeling assay. FSC, forward scatter. These data are as presented in Hodge et al. (([1999])).

Figure 5. C57BL//6 mice ((five//group)) were administered HBSS ((closed squares)) or vaccinated with 10⁷ pfu rV‐‐CEA ((closed triangles)) or rV‐‐CEA//TRICOM ((closed circle)). One hundred days later, mice were inoculated with 1 × 10⁶ MC38 carcinoma cells expressing CEA, and survival was monitored. All mice other than the rV‐‐CEA//TRICOM group developed tumors and were sacrificed when tumors exceeded 20 mm in length or width, or when the mice were moribund. Insert: In a second experiment, C57BL//6 mice ((five//group)) were vaccinated with 10⁷ pfu rV‐‐CEA, rV‐‐CEA//B7‐‐1, rV‐‐CEA//TRICOM or HBSS buffer. Lymphoproliferative responses from pooled splenic T cells were analyzed 22 days after vaccination. Values represent the stimulation index of the mean cpm of triplicate samples versus media. Standard deviation never exceeded 10%%. Antigens used were Con A ((5 μg//ml)), CEA ((100 μg//ml)), and ovalbumin ((100 μg//ml)). These data are as presented in Hodge et al. (([1999])).

Figure 6. Efficiency of poxviral infection of dendritic cells. Uninfected dendritic cells are depicted in panels A and C. Dendritic cells were infected with either a multiplicity of infection ((MOI)) of 50 plaque‐‐forming units per cell of rF‐‐CEA//TRICOM ((panel B)) or 25 MOI of rV‐‐TRICOM ((panel D)) for 5 h. After 18 h, cells were analyzed for surface co‐‐expression of CD11b and either carcinoembryonic antigen ((CEA)), a marker gene for rF‐‐CEA//TRICOM ((panels A and B)), or vaccinia proteins via polyclonal rabbit anti‐‐vaccinia serum ((panels C and D)). For panels A–D, the y‐‐axis represents the fluorescent intensity of cells expressing CD11b. For panels A and B, the x‐‐axis represents the fluorescent intensity of cells expressing CEA proteins. For panels C and D, the x‐‐axis represents the fluorescent intensity of cells expressing vaccinia proteins. The insert boxes in each panel denote the percent positive cells in each quadrant. Dendritic cells infected with TRICOM vectors exhibit enhanced capacity to stimulate naive T cells ((panels E and F)). All dendritic cell populations were co‐‐cultured for 48 h with T cells at a ratio of 10:1 in the presence of different concentrations of cancanavalin A ((Con A)) to provide signal 1. ³H‐‐thymidine was added during the final 18 h. Panel E: Uninfected dendritic cells ((open squares)), mock‐‐infected dendritic cells ((open diamonds)), or dendritic cells infected with wild‐‐type fowlpox ((FP‐‐WT)) ((open inverse triangles)), rF‐‐B7‐‐1 ((closed triangles)) or rF‐‐TRICOM ((closed circles)). Panel F: Dendritic cells ((open squares)), mock‐‐infected dendritic cells ((open diamonds)), or dendritic cells infected with wild‐‐type vaccinia ((V‐‐WT)) ((open inverse triangles)), rV‐‐B7‐‐1 ((closed triangles)) or rF‐‐TRICOM ((closed circles)). Error bars represent 95%% confidence intervals. In some cases, the error bars are obscured by the symbols. These data are as presented in Hodge et al. (([2000])).

Table 1. Infection of dendritic cells ((DCs)) with rV‐‐TRICOM or rF‐‐TRICOM: increase in the expression level of B7‐‐1, ICAM‐‐1, and LFA‐‐3.^a

Infection	DC panel, %% positive cells ((MFI))									Non‐‐DC markers %% positive cells ((MFI))
	I‐‐A^b	H‐‐2K^b//D^b	CD11b	CD11c	B7‐‐2	CD40	B7‐‐1	ICAM‐‐1	LFA‐‐3	CD‐‐14	CD‐‐19	CD3	Pan NK
None	88 ((1124))	89 ((125))	93 ((935))	20 ((74))	68 ((490))	68 ((82))	91 ((329))	96 ((595))	88 ((153))	2 ((20))	2 ((26))	0.3 ((33))	2 ((56))
Mock	87 ((989))	88 ((125))	90 ((1129))	25 ((49))	77 ((432))	71 ((99))	85 ((330))	97 ((519))	86 ((189))	1 ((42))	4 ((30))	0.6 ((37))	2 ((29))
FP‐‐WT	91 ((985))	98 ((126))	89 ((889))	24 ((69))	65 ((487))	71 ((90))	94 ((382))	97 ((464))	91 ((130))	2 ((18))	2 ((10))	1 ((60))	2 ((31))
rF‐‐B7‐‐1	88 ((987))	99 ((114))	86 ((793))	28 ((72))	74 ((404))	68 ((90))	95 ((1559))	98 ((437))	90 ((180))	2 ((30))	2 ((29))	1 ((45))	2 ((54))
rF‐‐TRICOM	90 ((900))	99 ((115))	84 ((789))	27 ((66))	76 ((499))	73 ((93))	99 ((1824))	98 ((1697))	95 ((530))	3 ((22))	3 ((13))	1 ((50))	3 ((33))
V‐‐WT	87 ((890))	86 ((83))	86 ((588))	29 ((54))	61 ((274))	79 ((98))	90 ((197))	95 ((241))	70 ((196))	3 ((20))	3 ((45))	1 ((59))	5 ((50))
rV‐‐B7‐‐1	85 ((856))	85 ((104))	81 ((693))	25 ((62))	73 ((304))	73((103))	94 ((1689))	97 ((364))	72 ((131))	3 ((42))	3 ((40))	0.8 ((67))	4 ((37))
rV‐‐TRICOM	87 ((901))	78 ((103))	77 ((558))	22 ((54))	66 ((298))	71 ((81))	96 ((1442))	94 ((1528))	92 ((304))	3 ((32))	4 ((29))	0.9 ((70))	4 ((31))

^aDendritic cells ((DC)) were uninfected, mock infected, or infected with a multiplicity of infection ((MOI)) of 50 plaque‐‐forming units per cell of either fowlpox wild‐‐type ((FP‐‐WT)), recombinant fowlpox B7‐‐1 ((rF‐‐B7‐‐1)), or rF‐‐TRICOM or 25 MOI of either vaccinia wild‐‐type ((V‐‐WT)), recombinant vaccinia B7‐‐1 ((rV‐‐B7‐‐1)), or rV‐‐TRICOM for 5 h. After an 18‐‐h incubation, cells were phenotyped by three‐‐color flow cytometry analysis. Bold numbers indicate a > 100%% change in cell‐‐surface expression ((mean fluorescent intensity; MFI)). Other abbreviations used: TRICOM, triad of costimulatory molecules ((B7‐‐1, ICAM‐‐1 [[intercellular adhesion molecule‐‐1]], and LFA‐‐3 [[i.e., leukocyte function‐‐associated antigen‐‐3]])); I‐‐A^b == major histocompatibility complex class II allele; H‐‐2K^b//D^b == major histocompatibility complex class I allelles; NK == natural killer cell.

These data are as presented in Hodge et al. (([2000])).

Figure 7. Effect of TRICOM vectors on the stimulation of effector T cells. Panels A, B: Effect of fowlpox virus infection of dendritic cells on OVA ((i.e., ovalbumin_257–264)) peptide‐‐specific T‐‐cell proliferation. Uninfected dendritic cells ((open squares)) or dendritic cells infected with wild‐‐type fowlpox ((FP‐‐WT)) ((open inverted triangles)), rF‐‐B7‐‐1 ((closed triangles)), or rF‐‐TRICOM ((closed circles)) were co‐‐cultured with OVA peptide‐‐specific T cells. Experimental conditions included a fixed effector to stimulator cell ratio of 10:1 in the presence of various concentrations of OVA peptide or negative control peptide VSVN ((i.e., vesicular stomatitis virus N_52–59)) ((open diamonds)) ((panel A)) or a fixed peptide concentration of 1 μM in the presence of various effector to stimulator cell ratios ((panel B)). Panels C and D: Effect of vaccinia virus infection of dendritic cells on peptide‐‐specific T‐‐cell proliferation. Uninfected dendritic cells ((open squares)) or dendritic cells infected with wild‐‐type vaccinia ((V‐‐WT, open inverted triangles)), rV‐‐B7‐‐1 ((closed triangles)), or rV‐‐TRICOM ((closed circles)) were co‐‐cultured with OVA peptide‐‐specific T cells. Experimental conditions included a fixed effector to stimulator cell ratio of 10:1 in the presence of various concentrations of OVA peptide or negative control peptide VSVN ((open diamonds)) ((panel C)), or a fixed peptide concentration of 1 μM in the presence of various effector to stimulator cell ratios ((open diamonds)) ((panel D)). Error bars represent 95%% confidence intervals. In some cases, the error bars are obscured by the symbols. These data are as presented in Hodge et al. (([2000])).

Figure 8. Enhancing effect of rF‐‐TRICOM or rV‐‐TRICOM infection on the T‐‐cell stimulatory capacity of dendritic cells of intermediate maturity ((granulocyte‐‐macrophage colony‐‐stimulating factor and interleukin 4, and noted as dendritic cells)) and further matured dendritic cells using tumor necrosis factor ((TNF))‐‐α, lipopolysaccharide ((LPS)), or CD40‐‐specific monoclonal antibody ((mAb)). Dendritic cells ((closed squares)) or dendritic cells matured with either 100 ng//mL TNF‐‐α ((open triangles)), 0.1 μg//mL LPS ((open diamonds)), or 5 μg//mL CD40‐‐specific mAb ((open circles)) for the final 24 h of culture were used to stimulate OVA ((ovalbumin_257–264))‐‐specific effector T cells in the presence of several concentrations of OVA peptide ((panel A)). Aliquots of all the above dendritic cell populations were then infected with a multiplicity of infection ((MOI)) of 50 plaque‐‐forming units per cell of rF‐‐TRICOM and used to stimulate OVA‐‐specific T cells under similar conditions ((panel B)). In a separate set of experiments, dendritic cells cultured with TNF‐‐α ((open triangles)), LPS ((open diamonds)), or CD40‐‐specific mAb ((open circles)) as above were used to stimulate OVA‐‐specific effector T cells in the presence of several concentrations of OVA peptide ((panel C)). Aliquots of all dendritic cell populations were then infected with 25 MOI of rV‐‐TRICOM and used to stimulate OVA‐‐specific T cells under similar conditions ((panel D)). For all panels, the T‐‐cell to dendritic cell ratio was 10:1, and the OVA peptide concentration was 1 μg//mL. Closed circles denote proliferation of OVA T cells stimulated with all dendritic cell populations in the presence of 1 μg//mL VSVN ((i.e., vesicular stomatitis virus N_52–59)) peptide. Error bars represent 95%% confidence intervals. In some cases, the error bars are obscured by the symbols. These data are as presented in Hodge et al. (([2000])).

Figure 9. Effect of poxvirus infection of dendritic cells on induction of cytotoxic T lymphocyte ((CTL)) activity ((panels A–J)). Groups of mice were vaccinated subcutaneously with 100 μg peptide in Ribi//Detox adjuvant ((peptide//adjuvant)) ((panels A and F)) for comparison with dendritic cell vaccination. Uninfected dendritic cells ((panels B and G)), dendritic cells infected with wild‐‐type fowlpox ((FP‐‐WT)) or wild‐‐type vaccinia ((V‐‐WT)) ((panels C and H)), dendritic cells infected with recombinant fowlpox or recombinant vaccinia expressing B7‐‐1 ((DC//B7‐‐1)) ((panels D and I)), dendritic cells infected with recombinant fowlpox or recombinant vaccinia expressing TRICOM ((DC//TRICOM)) ((panels E and J)) were pulsed with 10 μM OVA ((i.e., ovalbumin _257–264)) peptide for 2 h and administered intravenously to mice ((1 × 10⁵ cells//mouse)). Fourteen days later, the spleens were harvested, after which spleen cell suspensions were restimulated for 6 days with the corresponding peptide and assessed for lytic ability against EL‐‐4 cells pulsed with either OVA ((closed squares)) or VSVN ((i.e., vesicular stomatitis virus N_52–59)) peptide ((open squares)). Numbers in panels depict CTL activity as expressed in lytic units ((LU)), as calculated using the following formula: LU_18%% == [[((1 × 10⁶))//((5,000 × number of effector cells to reach 18%% lysis))]]. Error bars represent 95%% confidence intervals. In some cases, the error bars are obscured by the symbols. These data are as presented in Hodge et al. (([2000])).

Hodge J. W., Sabzevari H., Yafal A. G., Gritz L., Lorenz M. G.O., Schlom J. A triad of costimulatory molecules synergize to amplify T‐‐cell activation. Cancer Res. 1999; 59: 5800–5807

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Hodge J. W., Rad A. N., Grosenbach D. W., Sabzevari H., Yafal A. G., Gritz L., Schlom J. Enhanced activation of T cells by dendritic cells engineered to hyperexpress a triad of costimulatory molecules. J. Natl. Cancer Inst. 2000; 92: 1228–1239

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