Antibody association with HER-2/neu-targeted vaccine enhances CD8 T cell responses in mice through Fc-mediated activation of DCs

Abstract

The pathogenic nature of cancer is attributed, at least in part, to the ability of tumors cells to induce systemic and local mechanisms of immune tolerance. However, we previously reported that tumor-free survival in up to 100% of tolerized HER-2/neu transgenic mice can be achieved by administration of neu-specific mAb concurrently with a HER-2/neu-expressing, GM-CSF-secreting whole cell vaccine. In this report, we show that one mechanism of improved antitumor activity induced by the combination of these 2 neu-targeted interventions was enhanced Fc-mediated activation of APCs. Specifically, in vivo studies demonstrated localization of radiolabeled neu-specific mAb at the vaccine site. Subsequently, increased accumulation of neu-specific mAb at the vaccine-draining lymph node correlated with increased vaccine cell uptake by DCs in vivo. This led to enhancement of CD8(+) neu-specific T cell function in terms of proliferation, cytokine production, and central memory development. Thus, the administration of a neu-specific mAb with a neu-targeted GM-CSF-secreting tumor vaccine enhanced induction of neu-specific CD8(+) T cells through Fc-mediated activation of DCs. This multimodality attack on the same tumor antigen may have the potential to overcome tolerance to self antigens and weaken the immunosuppressive networks within the tumor microenvironment.

Figure 1. The Fc portion of the intact 7.16.4 mAb is required for enhancement of the vaccine-induced antitumor response.

(A) Binding of F(ab′)₂ fragments of 7.16.4 (dashed line) were confirmed by flow cytometry, using secondary antibodies specific for the Fc portion and λ₁, λ₂, and λ₃ light chains. Shown are the intact antibody (solid line) and control IgG (gray histogram). (B) Antibody-mediated enhancement of the vaccine-induced antitumor response is not seen in the vaccine + F(ab′)₂ treatment group. neu-N mice received neu-targeted vaccine (3T3 neu/GM) or control vaccine (3T3 NP/GM), followed 2 weeks later by NT2 challenge + intact 7.16.4 mAb, + 7.16.4 F(ab′)₂, + or irrelevant IgG. The intact and control antibodies were administered weekly for a total of 5 injections (100 μg of IgG per injection). F(ab′)₂ fragments were injected twice per week for a total of 10 injections [150 μg of F(ab′)₂ per injection]. Survival curves are shown (n = 5 per group). Statistical differences in survival among data groups were assessed using the log-rank test. This experiment was repeated once. (C) Tumor-specific CD8⁺ T cell responses were not increased in the vaccine + F(ab′)₂ treatment group when compared with the vaccine plus intact antibody treatment group. CD8⁺ T cells were incubated with NT2/B7-1 target cells overnight at 37°C at 5% CO₂. IFN-γ ELISPOTs were counted on the next day. Each experiment was repeated 3 times independently. *P < 0.05 versus intact 7.16.4 mAb + neu-targeted vaccine, Mann-Whitney U test.

Figure 2. Intact 7.16.4 mAb accumulates preferentially within the draining lymph nodes of neu-expressing, GM-CSF–secreting tumor vaccine cells.

(A) Indium-111–labeled intact 7.16.4 mAb forms a hot spot (arrow) at the location of the vaccine. neu-N mice were given the neu vaccine cells (3T3 neu/GM) (1 × 10⁶) in the right leg or the control GM-CSF–secreting vaccine cells (3T3 NP/GM) (1 × 10⁶) in the left leg s.c., concurrent with an i.v. injection of indium-111–labeled intact 7.16.4 mAb. The SPECT/CT images, using an XPECT system, were taken at 1, 24, and 48 hours. Three mice per group were analyzed per experiment, and these experiments were repeated once with similar results. (B) Accumulation of indium-111–labeled intact 7.16.4 mAb at the site of a neu-targeted vaccine and its draining lymph node. Administration of vaccine cells and antibodies was as same as in A. After 48 hours, VDLNs (left and right inguinal) and superficial cervical lymph nodes (used as a control for the non-draining lymph nodes) were isolated and their radioactivity was measured in a gamma counter. Plotted is the mean radioactivity (injection dose %) for 5 mice per group. P values comparing right and left inguinal lymph nodes were determined by Mann-Whitney U test.

Figure 3. CD11c⁺ DCs take up vaccine cells most efficiently in mice treated with the neu-expressing, GM-CSF–secreting vaccine given concurrently with the intact 7.16.4 mAb.

(A) neu-specific antibody increased the uptake of vaccine cells by CD11c⁺ DCs in an Fc-dependent manner. neu-targeted vaccine (3T3 neu/GM) or control vaccine (3T3/GM) cells were labeled with PKH67 and then injected s.c. (1 × 10⁶ cells) into each limb of the mouse, followed by an i.p. injection of intact 7.16.4 mAb, 7.16.4 F(ab′)₂, or a control IgG Ab. The VDLNs were harvested daily on days 1–5 following treatment, and DCs were isolated using the Miltenyi CD11c magnetic beads. Plotted are the mean percentages of PKH67-expressing CD11c⁺ DCs for 4–5 mice per group. A representative flow cytometric analysis is shown for the neu-targeted vaccine + intact 7.16.4 mAb, + 7.16.4 F(ab′)₂, and + mIgG groups. *P < 0.05 versus all other treatment groups, Mann-Whitney U test. The statistical analysis is shown in Table 1. (B) CD11c⁺ DCs isolated from the neu-targeted vaccine and intact 7.16.4 mAb–treated mice expressed higher levels of the maturation markers B7-1, B7-2, CD40, MHC-II, and PD-L1 when compared with CD11c⁺ DCs isolated from mice treated with the neu-targeted vaccine + 7.16.4 F(ab′)₂ or + control antibody. Shown is a representative flow cytometric analysis of CD11c⁺ DCs isolated from 1 mouse per group. This experiment was repeated 3 times with similar results.

Figure 4. CD11c⁺ DCs isolated from mice treated with the neu-expressing, GM-CSF–secreting vaccine given concurrently with intact 7.16.4 mAb induce efficient proliferation of naive neu-specific CD8⁺ T cells.

(A) Naive RNEU_420–429-specific CD8⁺ T cells proliferated most vigorously in response to CD11c⁺ DCs isolated from mice treated with the neu-expressing, GM-CSF–secreting vaccine + intact 7.16.4 mAb. Administration of vaccine cells and antibodies was similar to that described in Figure 3A. On day 4 after vaccination, the spleen and VDLNs were harvested and DCs were isolated using the Miltenyi CD11c magnetic beads. Naive RNEU_420–429-specific CD8⁺ T cells were isolated, labeled with CFSE, and then cocultured with CD11c⁺-isolated DCs for 3 days. CFSE dilution was measured by flow cytometry. n = 5 mice per group. This analysis was repeated at least twice. (B) Both CD8⁺ and CD8^– DCs proliferated RNEU_420–429-specific CD8⁺ T cells most efficiently when the intact 7.16.4 mAb was administered with the neu-targeted vaccine. Administration of vaccine cells and antibodies was similar to that described in Figure 3A. On day 4, splenic CD11c⁺ DCs were isolated from each group, followed by FACS of CD8⁺ and CD8^– DCs. Subsequently, the DEAD assay was performed as described in A. n = 10 mice per group. This analysis was repeated at least twice. *P < 0.05 versus intact 7.16.4 mAb + neu-targeted vaccine, Mann-Whitney U test.

Figure 5. The neu-expressing, GM-CSF–secreting vaccine given concurrently with the intact 7.16.4 mAb enhances the effector function and proliferation capability of neu-specific CD8⁺ T cells in vivo.

(A) The neu-expressing, GM-CSF–secreting vaccine given concurrently with the intact 7.16.4 mAb increases the number of activated IFN-γ–expressing RNEU_420–429-specific CD8⁺ T cells following treatment. Each neu-N mouse received 2 × 10⁶ Thy1.2 RNEU_420–429-specific CD8⁺ T cells i.v., followed by 1 × 10⁶ 3T3 neu/GM or 3T3/GM cells in each limb s.c. and intact 7.16.4 mAb (100 μg), 7.16.4 F(ab′)₂ (150 μg), or irrelevant IgG (100 μg) i.p. on day 0. Their spleens and VDLNs were harvested on day 4, and CD8⁺ T cells were isolated with the Miltenyi CD8a magnetic beads. The isolated CD8⁺ T cells were then cocultured (1 × 10⁶) with RNEU_420–429-pulsed T2D^q (1 × 10⁶) overnight. Thy1.2 RNEU_420–429-specific CD8⁺ T cells were stained for IFN-γ and analyzed by flow cytometry. The mean fluorescent intensity of IFN-γ in Thy1.2 RNEU_420–429-specific CD8⁺ T cells was also measured. Shown is a representative flow cytometric analysis for 1 mouse per group. This study was performed on a total of 3 mice per group per experiment and was repeated once. The statistical analysis is shown in Table 2. *P < 0.05 as determined by the Mann-Whitney U test compared with 3T3 neu/GM + intact 7.16.4 mAb group. (B) The intact 7.16.4 mAb enhances proliferation of adoptively transferred TCR transgenic T cells in vaccinated neu-N mice. CFSE dilution of Thy1.2 RNEU_420–429-specific CD8⁺ T cells was measured by flow cytometry. Shown is a representative flow cytometric analysis of 1 mouse per group. A total of 3 mice per group were analyzed per experiment, and this experiment was repeated once. The statistical analysis is shown in Table 3.

Figure 6. A neu-expressing, GM-CSF–secreting vaccine given concurrently with the intact 7.16.4 mAb promotes the development of RNEU_420–429-specific CD8⁺ T_CM.

(A) The intact 7.16.4 mAb increases the total population of RNEU_420–429-specific CD8⁺ T cells with time. Each neu-N mouse received the same treatment protocol as described in Figure 5A, except 4 × 10⁶ Thy1.2⁺ RNEU_420–429-specific CD8⁺ T cells were used instead of 2 × 10⁶ cells. After 30 days, the mice were boosted with 1 × 10⁶ 3T3 neu/GM cells in all 4 limbs. One week after the boost, their spleens and VDLNs were harvested and CD8⁺ T cells were isolated using the Miltenyi CD8a magnetic beads. The proportion of Thy1.2⁺ RNEU_420–429-specific CD8⁺ T cells present in the isolated CD8⁺ T cell population was analyzed by flow cytometry. (B) The intact 7.16.4 mAb enhances proliferation of RNEU_420–429-specific CD8⁺ T_CM. Thy1.2⁺ RNEU_420–429-specific CD8⁺ T cells from the experiment described in A were stained for CD62L and CD44 and analyzed by flow cytometry. Shown is a representative flow cytometric analysis of 1 mouse per group. A total of 3 mice per group were analyzed, and this study was repeated once. The gating represents Thy1.2⁺ CD8⁺ T cells. *P < 0.05 versus intact 7.16.4 mAb + neu-targeted vaccine, Mann-Whitney U test.