CD4+ Th1 cells promote CD8+ Tc1 cell survival, memory response, tumor localization and therapy by targeted delivery of interleukin 2 via acquired pMHC I complexes

Abstract

The cooperative role of CD4+ helper T (Th) cells has been reported for CD8+ cytotoxic T (Tc) cells in tumor eradication. However, its molecular mechanisms have not been well elucidated. We have recently demonstrated that CD4+ Th cells can acquire major histocompatibility complex/peptide I (pMHC I) complexes and costimulatory molecules by dendritic cell (DC) activation, and further stimulate naïve CD8+ T cell proliferation and activation. In this study, we used CD4+ Th1 and CD8+ Tc1 cells derived from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic OT II and OT I mice to study CD4+ Th1 cell's help effects on active CD8+ Tc1 cells and the molecular mechanisms involved in CD8+ Tc1-cell immunotherapy of OVA-expressing EG7 tumors. Our data showed that CD4+ Th1 cells with acquired pMHC I by OVA-pulsed DC (DCOVA) stimulation are capable of prolonging survival and reducing apoptosis formation of active CD8+ Tc1 cells in vitro, and promoting CD8+ Tc1 cell tumor localization and memory responses in vivo by 3-folds. A combined adoptive T-cell therapy of CD8+ Tc1 with CD4+ Th1 cells resulted in regression of well-established EG7 tumors (5 mm in diameter) in all 10/10 mice. The CD4+ Th1's help effect is mediated via the helper cytokine IL-2 specifically targeted to CD8+ Tc1 cells in vivo by acquired pMHC I complexes. Taken together, these results will have important implications for designing adoptive T-cell immunotherapy protocols in treatment of solid tumors.

Figure 1

Phenotypic characterization of in vitro DC_OVA-activated CD4⁺ Th1 and CD8⁺ Tc1 cells.(a) The in vitro DC_OVA-activated CD4⁺ Th1 and CD8⁺ Tc1 cells as described in Materials and Methods were stained using a panel of Abs for analysis of CD4, CD8, CD11c, CD25, CD69, CD80, Ia^b and Vβ5·1TCR (solid lines). The isotype-matched irrelevant Abs were used as controls (dotted lines).(b) The supernatants of these CD4⁺ Th1, Th1(pMHC I^–/–) and CD8⁺ Tc1 cells were assayed for IFN-γ, IL-4 and IL-2 secretion by ELISA, respectively. The values presented represent the means of triplicate cultures from three distinct experiments.(c) Transfer of H-2K^b and pMHC I molecules onto CD4⁺ Th1 cells by DC activation. The active CD4⁺ Th1 or CD4⁺ Th1(K^b–/–) cells (solid lines) derived from activation of naïve OT II CD4⁺ T cells or CD4⁺ T(K^b–/–) cells of OT II/H-2K^b–/– mice by DC_OVA, and the original naïve CD4⁺ T or CD4⁺ T(K^b–/–) cells (thick dotted lines) as well as the active CD4⁺ Th1(pMHC I^–/–) cells (solid lines) derived from activation of naïve OT II CD4⁺ T cells by (K^b–/–)DC_OVA, and the original naïve OT II CD4⁺ T cells (thick dotted lines) were stained with a panel of Abs for H-2K^b and pMHC I and analyzed by flow cytometry. The isotype-matched irrelevant Abs were used as controls (thin dotted lines). One representative experiment of three in the above different experiments is shown.

Figure 2

Functional characterization of in vitro DC_OVA-activated CD4⁺ OT II Th1 cells.(a) Naïve OT I CD8⁺ T cell proliferation assay. Varying numbers of stimulators including the irradiated CD4⁺ Th1, Th1(K^b–/–), Th1(pMHC I^–/–) cells and DC_OVA were cocultured with a constant number of naïve OT I CD8⁺ T cells. After two days, the proliferative responses of the CD8⁺ T cells were determined by ³H-thymidine uptake assays. (b) Apoptosis formation assay. Active CD8⁺ Tc1 cells were harvested 4 days after in vitro DC_OVA activation, and cultured with or without CD4⁺ Th1 cells in the medium without IL-2 for 4 days. T cells were harvested, stained with FITC-Annexin V and PE-anti-CD8 Ab, and analyzed by flow cytometry. In some experiments, each of a panel of neutralizing reagents (anti-IL-2 and -IFN-γ Abs) (each 15 µg/ml) was added to the culture system. Th1(pMHC I^–/–) cells without acquired pMHC I complexes were used as control cells in the above assay. *P < 0·05 (Student t-test) vs. cohorts of Th1 plus Tc1 cells. One representative experiment of two in the above different experiments is shown.

Figure 3

Enumeration of OVA-specific CD8⁺ Tc1 cells in tumors. Active CD8⁺ Tc1 cells (5 × 10⁶ cells/mouse) alone or together with in vitro-activated Th1 cells (2 × 10⁶ cells/mouse) were i.v. injected into EG7 tumor-bearing C57BL/6 mice.(a) Tumors were removed for preparation of cell suspensions as described in Materials and Methods at different days subsequent to T cell injection. T cells were purified from the cell suspensions by using anti-CD3 Ab-coated magnetic beads and analyzed by flow cytometry using PE-tetramer and FITC-labeled anti-CD8 Ab.(b) The tumor-bearing mice were injected i.v. with 5 × 10⁶ Tc1 cells alone or in conjunction with 2 × 10⁶ Th1 cells. Tumors were removed for preparation of cell suspansions 3 days after T cell injection. Plots display the percentages of FITC-CD8 and PE-tetramer positive CD8⁺ Tc1 cells in the total CD8⁺ T cell population.(c) The tumor-bearing mice were injected i.v. with 5 × 10⁶ Tc1 cells in conjunction with 2 × 10⁶ Th1, Th1(IL-2^–/–), Th1(IFN-γ^–/–), Th1(pMHC I^–/–) and Th1(K^b–/–) cells. Tumors were removed for preparation of cell suspensions 3 days after T cell injection. *P < 0·05 vs. cohorts of injection of Tc1 plus Th1 cells (Student's t-test). Data shown are representative of three separate experiments with three to five mice per experimental group.

Figure 4

Impact on tumor growth and mortality rates of cotransfer of Tc1 with Th1 cell subset in adoptive T-cell immunotherapy of established tumors. Mice bearing established (i.e. ∼5 mm in diameter) EG7 tumors were given i.v. injections of 5 × 10⁶ CD8⁺ Tc1 in conjuction with different amounts of CD4⁺ Th1 cells. (a) Tumor growth was monitored and the tumor size (diameter) measured daily using an engineering caliper. The evolution of the tumors in individual mice is depicted, as are the fractions of mice in each treatment group that were tumor-free at 60 days post-treatment. (b) The readout in this figure represents the long-term mortality among the animals, as determined by daily assessments across 60 days post-treatment. The data closely mirror that in (a) with a conclusion that the combined CD4⁺ Th1 and CD8⁺ Tc1 cell immunotherapy provides a significant therapeutic advantage over the single use of Tc1 cells alone. One representative experiment of two is shown.

Figure 5

Animal study using CD4⁺ Th1 cells with respective gene deficiency. The mice bearing tumors were injected i.v. with 5 × 10⁶ of Tc1 cells in conjunction with 2 × 10⁶ Th1, Th1(IL-2^–/–), Th1(IFN-γ^–/–), Th1(K^b–/–) and Th1(pMHC I^–/–) cells. Tumor growth was monitored and the tumor size (diameter) measured daily using an engineering caliper. The evolution of the tumors in individual mouse is depicted, as are the fractions of mice in each treatment group that were tumor-free at 60 days post-treatment.

Figure 6

CD4⁺ Th1 cells promote active CD8⁺ Tc1 cell memory responses.(a) In vitro DC_OVA-activated CD8⁺ Tc1 cells can become long-lived memory T cells. In vitro DC_OVA-activated CD8⁺ Tc1 cells (5 × 10⁶ cells/mouse) alone or in conjuction with active CD4⁺ Th1 or Th1(pMHC I^–/–) cells (2 × 10⁶ cells/mouse) were i.v. injected into C57BL/6 mice. Three months later, tail blood samples were taken from the mice, and a triple staining for PE-K^b/OVA tetramer, FITC-anti-CD8 and ECD-anti-CD44 Abs were conducted to determine the percentage of OVA-specific triple staining-positive CD8⁺ T cells in the total CD8⁺ population indicated in each right upper plot. The T cell population with PE-tetramer and FITC-CD8 double positivity was also the respective population with PE-tetramer and ECD-CD44 double positivity by flow cytometric analysis.(b) Three months later, the immunized mice were boosted by i.v. injection of 1 × 10⁶ irradiated DC_OVA. Four days after the boost, tail blood samples were taken from the boosted mice, and a double staining for PE-K^b/OVA tetramer and FITC-anti-CD8 Ab were conducted to determine the percentage of OVA-specific double staining-positive CD8⁺ T cells in the total CD8⁺ population indicated in each right upper plot. Naive C57BL/6 mice were also boosted with DC_OVA and used as control.(c) Three months later, the immunized mice were also s.c. challenged with 1 × 10⁶ or 3 × 10⁶ EG7 tumor cells. Mouse survival was monitored daily. The results presented are representative of two separate experiments with 4 mice (a & b) or 8 mice (c) per group.