Effect of B7.1 costimulation on T-cell based immunity against TAP-negative cancer can be facilitated by TAP1 expression

Abstract

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP(-)) or TAP1-transfected (TAP1(+)) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP(-) and TAP1(+) B7.1 expressing CMT.64 cells depending on the doses of gamma-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1(+) cells rejected TAP(-) tumors, only high dose immunization with B7.1-expressing TAP(-) cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP(-) tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of gamma-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP(-) cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.

Figure 1. Expression of TAP1, B7.1 and MHC-I molecules in transfectants.

TAP, B7.1, K^b and D^b expression in CMT.64 transfectants was determined. A) TAP1 and TAP2 proteins were detected in CMT.64 transfectants and control RMA cells by Western blots using anti-TAP1 and TAP2 polyclonal antibodies respectively (see Material and Methods). Levels of expression of GAPDH protein were detected in each sample as the loading control. B) B7.1 expression in CMT.B7.1/p and CMT.TAP1/B7.1 cells was detected by FACS assays using FITC-conjugated B7.1-specific mAb 16-10A1. Control is CMT.64 cells stained with mAb 16-10A1. C) K^b or D^b expression was detected by FACS assays using primary mAbs Y-3 against H-2K^b or 28-14-8S against H-2D^b followed by staining with a FITC-conjugated goat anti-mouse IgG secondary Ab. CMT.64 cells stained with a primary mAb 15-5-5S (against H-2D^k) followed by staining with a FITC-conjugated goat anti-mouse IgG secondary Ab were used as a negative control. a: negative control; b: CMT.64; c: CMT.64/pp; d: CMT.B7.1/p; e: CMT.TAP1/p; f: CMT.TAP1/B7.1; and g: CMT.TAP1,2 cl.21.

Figure 2. Decrease in tumorigenicity and increase in immune response by B7.1 and TAP1 co-expressing tumor cells.

The time of morbidity was recorded for mice (each group, n = 10) inoculated with live tumor cells or immunized with γ-irradiated cells and followed by challenge with CMT.64 cells. Statistics for mouse survival were obtained using the Kaplan–Meier log rank survival test and differences were considered significant at P<0.05. A) Tumorigenicity was detected by injection of C57BL/6 mice i.p. with live CMT.64 cells or their transfectants (5×10⁴ cells per mouse). B) Nude mice were used for determination of tumorigenicity with conditions of tumor injection the same as shown in A), P>0.05 for all comparisons. C) C57BL/6 mice were immunized i.p. with different γ-irradiated tumor cells or PBS at a high-dose (left, 5×10⁶ cells per mouse) or a low-dose (right, 5×10⁵ cells per mouse). After a 20-day immunization, the mice were challenged i.p. with live CMT.64 tumor cells (2.5×10⁵ cells per mouse). Different survival rates and/or times were observed.

Figure 3. Deduction or abolishment of immune response against CMT.64 tumor by depleting CD4⁺ or CD8⁺ T cell sub-population in C57BL/6 mice.

Mice (each group, n = 10) were injected i.p. with mAbs GK1.5 for CD4⁺ T cells or 2.43 for CD8⁺ T cells (0.1 ml per mouse) every other day for the first week and once per week afterward to deplete relevant T-cell sub-population. A) Before γ-irradiated tumor cell immunization, depletion of CD8⁺ or CD4⁺ T cell subsets was assessed in blood by FACS assays using FITC-conjugated anti-mouse CD8a (5H1-1) or FITC-conjugated anti-mouse CD4 (RM4-4) together with PE/Cy5-conjugated anti-mouse CD3 (145-2C11). Frequencies of CD8⁺ or CD4⁺ T cells were detected before mAb treatment (indicated as normal) and after first week mAb treatment and before γ-irradiated tumor cell immunization (indicated as mAb-treatment). B) After a 20-day immunization with γ-irradiated tumor cells (5×10⁶ cells per mouse) the mice were challenged i.p. with live CMT.64 tumor cells (2.5×10⁵ cells per mouse). The time of morbidity was recorded.

Figure 4. TAP-deficient tumor cells present TAP-independent antigen for T cell priming.

A) Left: RT-PCR was performed to detect two spliced (long and short) Lass5 gene transcripts in CMT.64, CMT.TAP1,2, cl.2 and RMA-S cells. Only the long transcript encodes a Lass5 epitope . A) Right: Lass5 epitope presentation by TAP-deficient and TAP-proficient CMT.64 cells was detected by 12–16 h ⁵¹Cr-release assays. Lass5 specific T cells were generated by immunization i.p with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM Lass5 peptide. The immunized splenocytes were re-stimulated in vitro with Lass5 peptide-pulsed γ-irradiated RMA-S/B7.1 cells for 5 days. One of three experiments is shown. B) Left: 12–16 h ⁵¹Cr-release assays were conducted to confirm that an NP_366–374 eptitope specific T-cell population generated by immunization with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM NP_366–374 peptide did not contain T-cell sub-populations recognizing TAP-independent epitopes presented by CMT.64 and its transfectants. The ⁵¹Cr-labled targets were shown in figure 4B. B) Right: Standard ⁵¹Cr-release assays were conducted to confirm that TAP-deficient CMT.64 transfectants did not present TAP-dependent NP_366–374 epitope when the cells were infected overnight with VV carrying NP_366–374 minigene at multiplicity of infection (MOI) of 3. The ⁵¹Cr-labled targets were shown in figure 4B. The NP_366–374 epitope specific T-cells were generated by immunization with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM NP_366–374 peptide and re-stimulated with 5 µM NP_366–374 peptide in vitro. C) An ELISPOT assay was performed to detect Lass5 specific IFN-γ-secreting precursors. Mice were immunized with γ-irradiated CMT.64/pp, CMT.TAP1,2, CMT.B7.1/p and CMT.TAP1/B7.1 tumor cells. The immunized splenocytes were stimulated with or without Lass5 peptide. The number of Lass5 antigen-specific, IFN-γ-secreting precursors was determined. Precursor frequency is reported as IFN-γ-secreting cells per 10⁶ splenocytes (IFN-γ-SC/10⁶ splenocytes).

Figure 5. Influence of TAP-independent tumor antigen presentation in virally infected TAP-deficient tumor cells.

CMT.64 cells were infected overnight with a combination of two VVs at MOI of 3 for each VV. A) B7.1 expression was detected by FACS assay using FITC-conjugated B7.1-specific mAb 16-10A1. CMT.64 cells without viral infection were used as a negative control. TAP1 expression was detected by Western blot using a goat anti-mouse TAP1 polyclonal Ab. RMA-S cells were used as a control. B) Standard ⁵¹Cr-release assays were conducted to detect if virally infected tumor cells affected presentation of endogenous tumor antigens. CMT.64 tumor cells were infected overnight with VV-GFP+VV-GFP, VV-B7.1+VV-GFP, or VV-B7.1+VV-TAP1, and used as target cells. Control cell lines were CMT.64, CMT.B7.1/p and CMT.TAP1/B7.1. Tumor antigen-specific T cells were generated by immunization with γ-irradiated CMT.B7.1/p cells. Target and effector ratio was used at 1∶100. a: CMT.64; b: CMT.B7.1/p; c: CMT.TAP1/B7.1; d: CMT.64+VV-GFP+VV-GFP; e: CMT.64+VV-B7.1+VV-GFP and f: CMT.64+VV-B7.1+VV-TAP1. ** indicates statistical significance (P≪0.05) using ANOVA analysis.