Activation of CD8⁺ T Cell Responses after Melanoma Antigen Targeting to CD169⁺ Antigen Presenting Cells in Mice and Humans

Abstract

The lack of tumor-reactive T cells is one reason why immune checkpoint inhibitor therapies still fail in a significant proportion of melanoma patients. A vaccination that induces melanoma-specific T cells could potentially enhance the efficacy of immune checkpoint inhibitors. Here, we describe a vaccination strategy in which melanoma antigens are targeted to mouse and human CD169 and thereby induce strong melanoma antigen-specific T cell responses. CD169 is a sialic acid receptor expressed on a subset of mouse splenic macrophages that captures antigen from the blood and transfers it to dendritic cells (DCs). In human and mouse spleen, we detected CD169⁺ cells at an equivalent location using immunofluorescence microscopy. Immunization with melanoma antigens conjugated to antibodies (Abs) specific for mouse CD169 efficiently induced gp100 and Trp2-specific T cell responses in mice. In HLA-A2.1 transgenic mice targeting of the human MART-1 peptide to CD169 induced strong MART-1-specific HLA-A2.1-restricted T cell responses. Human gp100 peptide conjugated to Abs specific for human CD169 bound to CD169-expressing monocyte-derived DCs (MoDCs) and resulted in activation of gp100-specific T cells. Together, these data indicate that Ab-mediated antigen targeting to CD169 is a potential strategy for the induction of melanoma-specific T cell responses in mice and in humans.

Keywords: CD169; Siglec-1; T cell responses; cancer vaccines; dendritic cell; macrophage; melanoma; sialoadhesin.

Figure 1

Targeting melanoma Ag to CD169 results in specific T cell responses in mice. Intravenous immunization with 1 µg Ab:Ag conjugates in the presence of 25 µg anti-CD40 Ab and 25 µg Poly(I:C). (A) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with Trp2 peptide 7 days after immunization with Trp2:Ab conjugates. (B–C) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with (B) human gp100 peptide or (C) mouse gp100 peptide 7 days after immunization with human gp100:Ab conjugates. (D) Percentage of CD8⁺ T cells binding H2-D^b tetramers 7 days after immunization with human gp100:Ab conjugates. Combined data of two experiments with 4–6 mice per group with one representative dotplot of each group is shown for all figures except for C which is one experiment with 6 mice per group. Statistical analysis one-way ANOVA with Sidak’s multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 1

Targeting melanoma Ag to CD169 results in specific T cell responses in mice. Intravenous immunization with 1 µg Ab:Ag conjugates in the presence of 25 µg anti-CD40 Ab and 25 µg Poly(I:C). (A) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with Trp2 peptide 7 days after immunization with Trp2:Ab conjugates. (B–C) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with (B) human gp100 peptide or (C) mouse gp100 peptide 7 days after immunization with human gp100:Ab conjugates. (D) Percentage of CD8⁺ T cells binding H2-D^b tetramers 7 days after immunization with human gp100:Ab conjugates. Combined data of two experiments with 4–6 mice per group with one representative dotplot of each group is shown for all figures except for C which is one experiment with 6 mice per group. Statistical analysis one-way ANOVA with Sidak’s multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Targeting HLA-A2.1 restricted Ag to CD169 results in Ag-specific T cell responses in HLA A2.1 transgenic mice. Intravenous immunization with 1 µg Ab:Ag conjugates in the presence of 25 µg anti-CD40 Ab and 25 µg Poly(I:C). (A) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with MART-1 peptide (B) Percentage of CD8⁺ T cells binding HLA-A2.1 tetramers 7 days after immunization with MART-1:Ab conjugates in HLA-A2.1 transgenic mice. Combined data of two experiments with 3–6 mice per group with one representative dotplot of each group is shown. Statistical analysis one-way ANOVA with Sidak’s multiple comparison test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

CD169 expression in mouse and human spleen. (A) Mouse spleen overview section stained for IgD (blue) and CD169 (red). (B) Human spleen overview section stained for CD19 (blue) and CD169 (red). (C) B cell follicles (blue) in human spleen from three different donors, CD169⁺ cells (red) and DC-SIGN⁺ cells (green). The first panel is a zoom-in from (B). (D) B cell follicles (blue) in human spleen from three different donors, CD169⁺ cells (red) and CD163⁺ cells (green). (E) zoom-in from first panel of (C).

Figure 4

Expression of CD169 and DC-SIGN on human MoDCs and binding and internalization of Ab-Ag conjugates. (A) Expression of DC maturation markers and CD169 on unstimulated MoDCs (black) or IFNγ stimulated MoDCs (grey) (n = 5). (B) Binding and uptake of specific Abs for CD169 (red), DC-SIGN (blue) and an isotype control (green). Cells were incubated with Abs for 45 min on ice, directly followed by 45 min incubation on ice with a secondary Ab. Subsequently, cells were kept on ice (t = 0 min), or incubated at 37 °C for indicated times and finally analyzed by flow cytometry (n = 4). (C) Receptor internalization after incubation with specific Abs for CD169 (red), DC-SIGN (blue) and an isotype control (green). Cells were incubated with the Abs for 45 min on ice and subsequently washed to remove unbound Ab. Next, cells were kept on ice (t = 0 min) or incubated at 37 °C for indicated times. Receptors remaining on the cell surface were visualized by flow cytometry after incubation with a secondary Ab (n = 3). (A–C) Representative histograms of DC-SIGN (blue, middle panel) and CD169 (red, right panel) expression are shown. (B,C) MFIs are normalized to isotype control at 0 min.

Figure 4

Expression of CD169 and DC-SIGN on human MoDCs and binding and internalization of Ab-Ag conjugates. (A) Expression of DC maturation markers and CD169 on unstimulated MoDCs (black) or IFNγ stimulated MoDCs (grey) (n = 5). (B) Binding and uptake of specific Abs for CD169 (red), DC-SIGN (blue) and an isotype control (green). Cells were incubated with Abs for 45 min on ice, directly followed by 45 min incubation on ice with a secondary Ab. Subsequently, cells were kept on ice (t = 0 min), or incubated at 37 °C for indicated times and finally analyzed by flow cytometry (n = 4). (C) Receptor internalization after incubation with specific Abs for CD169 (red), DC-SIGN (blue) and an isotype control (green). Cells were incubated with the Abs for 45 min on ice and subsequently washed to remove unbound Ab. Next, cells were kept on ice (t = 0 min) or incubated at 37 °C for indicated times. Receptors remaining on the cell surface were visualized by flow cytometry after incubation with a secondary Ab (n = 3). (A–C) Representative histograms of DC-SIGN (blue, middle panel) and CD169 (red, right panel) expression are shown. (B,C) MFIs are normalized to isotype control at 0 min.

Figure 5

Targeting melanoma Ag to CD169 on human MoDCs results in melanoma Ag presentation to T cells. IFNγ production by gp100_280–288-specific HLA-A2.1 restricted T cell line after 24 h co-culture with MoDCs previously pulsed for 3 h with Ab:gp100 c onjugates specific for CD169, DC-SIGN or isotype control Ab (n = 5 healthy donors, each different symbol represents one donor). Statistical analysis matched one-way ANOVA with Sidak’s multiple comparison correction * p < 0.05, ** p < 0.01.