Priming of immune responses against transporter associated with antigen processing (TAP)-deficient tumours: tumour direct priming

Abstract

We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2K(b) genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2K(b) gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2K(b) and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells.

Figure 1

Increased survival in tumour-bearing mice treated with transporter associated with antigen processing 1 (TAP1)- and B7.1-expressing tumour cells. C57BL/6 mice were inoculated with live CMT.64 cells (1 × 10⁵ cells per mouse). Three and six days after live tumour cell inoculation, mice (n = 10 in each group) were treated intraperitoneally (i.p.) with γ-irradiated CMT.TAP1/pEF4 tumour cells (1 × 10⁷ cells per mouse) that had been infected either with 1 : 1 [multiplicity of infection (MOI)] vaccinia virus (VV)-B7.1 and VV-K^b or with 1 : 1 (MOI) VV-GFP, and the time of morbidity was recorded. Mice treated with γ-irradiated CMT.64/pp₁ cells infected with 1 : 1 (MOI) VV-GFP were used as a negative control (P < 0·05 for mice immunized with CMT.TAP1/pEF4 cells infected with VV-B7.1 compared with mice immunized with CMT.TAP1/pEF4 cells infected with VV-GFP; P < 0·05 for mice immunized with CMT.TAP1/pEF4 cells infected with VV-GFP compared with mice immunized with CMT.64/pp₁ cells infected with VV-GFP).

Figure 2

Immunization with tumour cells infected with vaccinia virus (VV)-B7.1 + VV-transporter associated with antigen processing 1 (TAP1) increases survival in mice challenged with CMT.64 cells. C57BL/6 mice (n = 10 in each group) were inoculated intraperitoneally (i.p.) with 5 × 10⁶ cells/mouse γ-irradiated CMT.64 cells that had been infected with 1 : 3 [multiplicity of infection (MOI)] VV-GFP + 1 : 3 (MOI) VV-GFP, 1 : 3 (MOI) VV-TAP1 + 1 : 3 (MOI) VV-GFP or 1 : 3 (MOI) VV-B7.1 + 1 : 3 (MOI) VV-TAP1. After a 20-day immunization, mice were challenged i.p. with 2·5 × 10⁵ CMT.64 cells and survival was recorded (P < 0·05 for mice immunized with CMT.64 cells infected with VV-B7.1 + VV-TAP1 compared with mice immunized with CMT.64 cells infected with VV-GFP + VV-GFP; P > 0·05 for mice immunized with CMT.64 cells infected with VV-TAP1 + VV-GFP compared with mice immunized with CMT.64 cells infected with VV-GFP + VV-GFP).

Figure 3

Splenocytes immunized with transporter associated with antigen processing 1 (TAP1) and B7.1 co-expressing tumour cells increased interferon (IFN)-γ production. C57BL/6 mice (n = 3) were injected intraperitoneally (i.p.) with γ-irradiated tumour cells (5 × 10⁶ cells per mouse). Seven days after immunization, the splenocytes were stimulated with γ-irradiated CMT.64 cells (which were treated with 30 μg/ml mitomycin-c) at a ratio of 1 : 7 (tumour cells:spleenocytes). Supernatants of the culture were collected at day 5 after in vitro re-stimulation. The levels of secreted IFN-γ were quantified. The mean value of the results for three mice is shown. Statistical significance: **P < 0·0001; *P < 0·005.

Figure 4

Different recognition of transporter associated with antigen processing (TAP)-deficient CMT.64 cells by T cells generated by CMT.TAP1/B7.1 cells and by CMT.TAP1/B7.1 cell-lysate-loaded dendritic cells (DCs). Standard ⁵¹Cr-release assays were performed using major histocompatibility complex class I (MHC-I) mismatched Mid-T2 (negative control), CMT.64/pp and CMT.TAP1,2 cl.21 cells as targets. (a) Splenocyte-derived T cells were generated as follows. Bone marrow-derived DCs were loaded with lysates of γ-irradiated CMT.TAP1/B7.1 cells for 6 hr, followed by addition of lipopolysaccharide (LPS) for DC maturation. After washing, DCs were injected intraperitoneally (i.p.) into C57BL/6 mice (1 × 10⁶ cells per mouse) for immunization. After a 7-day immunization, the splenocytes were collected and re-stimulated with CMT.TAP1/B7.1 cell-lysate-loaded DCs for 5 days. (b) Splenocyte-derived T cells were generated by immunization of mice with γ-irradiated CMT.TAP1/B7.1 cells (5 × 10⁶ cells/mouse). After a 7-day immunization, the splenocytes were re-stimulated in vitro with γ-irradiated and mitomycin-c-treated CMT.TAP1/B7.1 cells for 4–5 days. One of three experiments is shown.