Comparison of Protein and Peptide Targeting for the Development of a CD169-Based Vaccination Strategy Against Melanoma

Abstract

CD169⁺ macrophages are part of the innate immune system and capture pathogens that enter secondary lymphoid organs such as the spleen and the lymph nodes. Their strategic location in the marginal zone of the spleen and the subcapsular sinus in the lymph node enables them to capture antigens from the blood and the lymph respectively. Interestingly, these specific CD169⁺ macrophages do not destroy the antigens they obtain, but instead, transfer it to B cells and dendritic cells (DCs) which facilitates the induction of strong adaptive immune responses. This latter characteristic of the CD169⁺ macrophages can be exploited by specifically targeting tumor antigens to CD169⁺ macrophages for the induction of specific T cell immunity. In the current study we target protein and peptide antigen as antibody-antigen conjugates to CD169⁺ macrophages. We monitored the primary, memory, and recall T cell responses and evaluated the anti-tumor immune responses after immunization. In conclusion, both protein and peptide targeting to CD169 resulted in strong primary, memory, and recall T cell responses and protective immunity against melanoma, which indicates that both forms of antigen can be further explored as anti-cancer vaccination strategy.

Keywords: CD169; Siglec-1; T cell; antigen; cross-presentation; macrophage; sialoadhesin; tumor immunology.

Figure 1

Targeting of protein and peptide to CD169 results in strong primary T cell responses. (A) Percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with SIINFEKL 7 days after immunization. (B) Percentage of CD8⁺ T cells binding H-2Kb-SIINFEKL tetramers. (C) same as in A in WT and Batf3 KO mice (D) Percentage of IFNγ producing CD4⁺ T cells after o.n. in vitro restimulation with I-Ab-restricted OVA_262−276. (E) Percentage of OVA-binding germinal center (GL7^hiCD38⁻) B cells (F) Relative percentage of IFNγ producing CD8⁺ T cells after 5 h in vitro restimulation with different concentrations of SIINFEKL. 100% is the IFNγ production after restimulation with the highest SIINFEKL concentration. (A–F) Splenocytes were taken 7 days after immunization with 1 μg Ab:Ag conjugates in the presence of 25 μg anti-CD40 Ab and 25 μg Poly(I:C). 1 representative experiment of 2–3 experiments is shown with 4–6 mice per group with one representative dotplot of each group. Statistical analysis one-way ANOVA with bonferroni's multiple comparison test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

Targeting of protein and peptide to CD169 results in long-lasting T cell responses. (A) Percentage of IFNγ producing CD8⁺ T cells (left panel) and IFNγ single and IFNγ /IL-2 double producing CD8⁺ T cells (right panel) after 5 h in vitro restimulation with SIINFEKL 28 days after immunization. (B) Percentage of CD8⁺ T cells binding H-2Kb-SIINFEKL tetramers 28 days after immunization. (C) Percentage of IFNγ single and IFNγ /IL-2 double producing CD8⁺ T cells after 5 h in vitro restimulation with SIINFEKL 7 days after immunization. (D) Percentage of central memory (CD44⁺CD62L⁺) and effector (CD44⁺CD62L⁻) antigen-specific CD8⁺ T cells at day 7 after immunization and representative dotplots for Tet⁺ and Tet⁻ CD8⁺ T cells are depicted. (A–D) 1 representative experiment of 2 is shown with 6 mice per group with a representative dotplot for each group. Statistical analysis one-way ANOVA with bonferroni's multiple comparison test ***p < 0.001.

Figure 3

Memory CD8⁺ T cell responses after targeting antigen to CD169. Mice were initially immunized with indicated Ab:Ag conjugates and the immune response was boosted 28 days later with 1 μg free SIINFEKL peptide in the presence of adjuvants, 7 days after boost splenocytes were used for analysis. (A) Percentage of IFNγ producing CD8⁺ T cells (left panel) and of IFNγ single and IFNγ/IL-2 double producing CD8⁺ T cells (right panel) after 5 h in vitro restimulation with SIINFEKL peptide. (B) Percentage of CD8⁺ T cells binding H-2Kb-SIINFEKL tetramers 28 days after immunization. (C) Percentage of central memory (CD44⁺CD62L⁺) and effector (CD44⁺CD62L⁻) of tetramer⁺ CD8⁺ T cells and representative dotplots for Tet⁺ and Tet⁻ CD8⁺ T cells are depicted. (A–C) Combined results of 2 experiments is shown with 4–6 mice per group with a representative dotplot for each group. Statistical analysis one-way ANOVA with bonferroni's multiple comparison test **p < 0.01.

Figure 4

Targeting of protein and peptide to CD169 results in the induction of tumor reactive T cell responses. (A–E) Mice were inoculated with 200,000 B16OVA tumor cells at day 0, on day 3 mice were immunized with indicated Ab:Ag conjugates in the presence of anti-CD40 Ab and Poly(I:C). Tumor size was monitored three times a week and mice were sacrificed based on physical appearance or tumor size. (A) Tumor size on different days after tumor inoculation, mean ± SEM is shown. Two-way ANOVA with Bonferroni correction comparing all groups on the last day of the experiment, only significant differences are depicted. (B) Tumor size per group of each mouse after tumor inoculation (C) Percentage of surviving mice on indicated days after tumor inoculation. (D) Tumor volume as in (A), but only showing the groups treated with the anti-CD169:Ag conjugates on a fitting scale. (E) Percentage of CD8⁺ T cells binding H-2Kb-SIINFEKL tetramers at indicated time points during the tumor experiment. (A–E) One experiment with 11 mice per group is shown. (F) Same as in (A), with treatment on day 7 after tumor cell inoculation, when all mice had a visible tumor. Data of one experiment with 11–12 mice per group is shown. Statistical analysis one-way ANOVA with bonferroni's multiple comparison test ***p < 0.001, ****p < 0.0001.