MHC class II and CD80 tumor cell-based vaccines are potent activators of type 1 CD4+ T lymphocytes provided they do not coexpress invariant chain

Abstract

We are developing vaccines that activate tumor-specific CD4+ T cells. The cell-based vaccines consist of MHC class I+ tumor cells that are genetically modified to express syngeneic MHC class II and costimulatory molecules. Previous studies demonstrated that treatment of mice with established tumors with these vaccines resulted in regression of solid tumors, reduction of metastatic disease, and increased survival time. Optimal vaccines will prime naïve T cells and activate T cells to tumor peptides derived from diverse subcellular compartments, since potential tumor antigens may reside in unique cellular locales. To determine if the MHC class II/costimulatory molecule vaccines fulfill these conditions, the vaccines have been tested for their ability to activate antigen-specific, naïve, transgenic CD4+ T lymphocytes. MHC class II(+)CD80+ vaccine cells were transfected with hen eggwhite lysozyme targeted to the cytosol, nuclei, mitochondria, or endoplasmic reticulum, and used as antigen-presenting cells to activate I-Ak-restricted, lysozyme-specific CD4+ 3A9 transgenic T cells. Regardless of the cellular location of lysozyme, the vaccines stimulated release of high levels of IFN-gamma and IL-2. If the vaccines coexpressed the MHC class II accessory molecule invariant chain, then IFN-gamma and IL-2 release was significantly reduced. These studies demonstrate that in the absence of invariant chain the MHC class II and CD80 tumor cell vaccines (1) function as antigen-presenting cells to activate naïve, tumor-specific CD4+ cells to endogenously synthesized tumor antigens; (2) polarize the activated CD4+ T cells toward a type 1 response; and (3) present epitopes derived from varied subcellular locales.

Fig. 1

SaI transfectants and transductants express MHC class II I-A^k, CD80, and/or HEL targeted to various subcellular compartments (ER, mitochondria, nuclei, cytoplasm). Transfectants and transductants were stained by indirect immunofluorescence for plasma membrane I-A^k (mAb 10–2.16), CD80 (mAb 1G10), or internal HEL (mAb HyHEL 7). Grey peaks denote staining with fluorescent conjugate without primary antibody; white peaks represent staining with primary antibody plus fluorescent conjugate. Numbers in the upper right-hand corner of each profile are the mean channel fluorescence for the antibody-stained peak. These data are representative of three independent experiments

Fig. 2

Activation of naïve, antigen (HEL)-specific CD4⁺ T cells to endogenously synthesized tumor antigen requires expression of MHC class II, HEL, and CD80 by the same tumor cells. Tumor cell transfectants expressing I-A^k and/or endogenously synthesized HEL (erHEL), and/or CD80/CD86 were incubated with I-A^k–restricted HEL-specific (HEL_46–61) 3A9 splenocytes. IL-2 release (pg/ml) was measured by ELISA. CD4⁺ T-cell activation occurs only if the stimulating tumor cells coexpress MHC class II, HEL, and CD80. These data are representative of four independent experiments

Fig. 3A–C

MHC class II⁺Ii⁻CD80⁺ SaI cells containing endogenously synthesized HEL localized to various subcellular compartments (SaI/A^k/B7/HEL) induce a strong type 1 response from naïve, HEL-specific, CD4⁺ T cells, while MHC class II⁺Ii⁺CD80⁺ SaI transfectants (SaI/CIITA/B7/HEL) are less effective APCs. Assorted SaI tumor cell transfectants synthesizing HEL localized to the ER, nuclei, cytoplasm, or mitochondria were incubated with naïve, adherent cell and B-cell-depleted 3A9 splenocytes at a ratio of 1:32, APCs to T cells. Cytokines were measured by ELISA: A IL-2 (pg/ml), B IFN-γ (ng/ml), C IL-4 (pg/ml). These data are representative of eight independent experiments

Fig. 4

SaI transfectants and transductants express MHC class II I-A^k, CD80, Ii, and/or HEL targeted to various subcellular compartments (ER, mitochondria, nuclei, cytoplasm). Transfectants and transductants were stained by indirect immunofluorescence for plasma membrane I-A^k‘ (mAb 10-2.16), CD80 (mAb 1G10), internal HEL (mAb HyHEL 7), or internal Ii (mAb In-1). Grey peaks denote staining with fluorescent conjugate without primary antibody; white peaks represent staining with primary antibody plus fluorescent conjugate. Numbers in the upper right-hand corner of each profile are the mean channel fluorescence for the antibody-stained peak. These data are representative of three independent experiments

Fig. 5A–C

Activation of naïve, antigen (HEL)-specific CD4⁺ T cells to exogenously synthesized tumor antigen is not affected by tumor cell expression of Ii or Ii plus DM. SaI transfectants or transductants were cocultured with exogenous HEL and adherent cell–depleted and B-cell–depleted 3A9 T cells at a ratio of 1:32, APCs to T cells. Cytokine release was measured by ELISA. A IL-2 (pg/ml), B IFN-γ (ng/ml), C IL-4 (pg/ml). These data are representative of four independent experiments