Analysis of CVC1302-Mediated Enhancement of Monocyte Recruitment in Inducing Immune Responses

Abstract

Monocytes (Mos) are believed to play important roles during the generation of immune response. In our previous study, CVC1302, a complex of PRRs agonists, was demonstrated to recruit Mo into lymph nodes (LNs) in order to present antigen and secret chemokines (CXCL9 and CXCL10), which attracted antigen-specific CD4⁺ T cells. As it is known that Mos in mice are divided into two main Mo subsets (Ly6C⁺ Mo and Ly6C^- Mo), we aimed to clarify the CVC1302-recruiting Mo subset and functions in the establishment of immunity. In this study, we found that CVC1302 attracted both Ly6C⁺ Mo and Ly6C^- Mo into draining LNs, which infiltrated from different origins, injection muscles and high endothelial venule (HEV), respectively. We also found that the numbers of OVA⁺ Ly6C⁺ Mo in the draining LNs were significantly higher compared with OVA⁺ Ly6C^- Mo. However, the levels of CXCL9 and CXCL10 produced by Ly6C^- Mo were significantly higher than Ly6C⁺ Mo, which plays important roles in attracting antigen-specific CD4⁺ T cells. Under the analysis of their functions in initiating immune responses, we found that the ability of the Ly6C⁺ monocyte was mainly capturing and presenting antigens, otherwise; the ability of the Ly6C^- monocyte was mainly secreting CXCL9 and CXCL10, which attracted antigen-specific CD4⁺ T cells through CXCR3. These results will provide new insights into the development of new immunopotentiators and vaccines.

Keywords: CVC1302; CXCL10; CXCL9; antigen; monocyte.

Figure 1

Kinetics of Ly6C⁺ Mo and Ly6C⁻ Mo recruitment into the draining LNs in responses to immunization with OVAF-CVC1302-ISA206 or OVAF-ISA206. BALB/c mice (twelve mice in each group) were immunized with OVAF-ISA206 or OVAF-CVC1302-ISA206. Popliteal LNs were harvested at 1, 3, 5 and 7 dpi and the numbers of targeted cells were evaluated with flow cytometry. (A) Representative flow cytometry plot showing OVA⁺ Ly6C⁺ Mo and OVA⁺ Ly6C⁻ Mo. (B) The numbers of total Ly6C⁺ Mo (left) and total Ly6C⁻ Mo (right) in draining LNs at 1, 3, 5 and 7 dpi. (C) The numbers of OVA⁺ Ly6C⁺ Mo (left) and OVA⁺ Ly6C⁻ Mo (right) in draining LNs at 1, 3, 5 and 7 dpi. (D) The percentages of OVA⁺ Ly6C⁺ Mo and OVA⁺ Ly6C⁻ Mo in OVA⁺ cells in mice immunized with OVAF-CVC1302-ISA206 (left) and OVAF-ISA206 (right). Data are presented as mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.0001.

Figure 2

The distribution pattern of Ly6C⁺ Mo and Ly6C⁻ Mo in draining LNs in response to immunization with OVA-CVC1302-ISA206. Mice were immunized intramuscularly with OVA-CVC1302-ISA206 and LNs were sampled at 3 dpi to analyze the distribution of Ly6C⁺ Mo and Ly6C⁻ Mo by confocal microscopy. LNs were stained with anti-Ly6C (red) and anti-CX3CR1 (green).

Figure 3

The origins of Ly6C⁺ Mo and Ly6C⁻ Mo infiltrated into the draining LNs in response to immunization with OVA-CVC1302-ISA206. BALB/c mice (twelve mice in each group) were immunized in the ear with OVA-CVC1302-ISA206, one group of mice were ear excised 2 h after immunization. Popliteal LNs were harvested at 1, 3, 5 and 7 dpi and stained with specific fluorescent antibody, then analyzed by flow cytometry. The numbers of total Ly6C⁺ Mo (A) and total Ly6C⁻ Mo (B) at the indicated time-points post-immunization. Data are presented as mean ± SEM; **** p ≤ 0.0001.

Figure 4

The potential of Ly6C⁺ Mo and Ly6C⁻ Mo in releasing CXCL9 and CXCL10 in response to immunization with OVA-CVC1302-ISA206. LNs were sampled from mice, immunized with OVA-CVC1302-ISA206, at 3 dpi to analyze the percentage of CXCL9 or CXCL10-producing Ly6C⁺ Mo and Ly6C⁻ Mo. (A) (left) Representative plot of CXCL9⁺ cells at 3 dpi. (right) MFI of CXCL9 expression with Ly6C⁺ Mo and Ly6C⁻ Mo. (B) (left) Representative plot of CXCL10⁺ cells at 3 dpi. (right) MFI of CXCL10 expression with Ly6C⁺ Mo and Ly6C⁻ Mo. (C) The distribution of CXCL9 or CXCL10-producing cells. Popliteal LNs were harvested at 3 dpi from mice immunized with OVA-CVC1302-ISA206. Cryosections (6 μm) of LNs were stained with anti-CXCL9 and anti-CXCL10, then visualized using confocal microscopy. Data are presented as mean ± SEM; * p ≤ 0.05, *** p ≤ 0.0001.

Figure 5

The distribution of antigen-specific CD4⁺ T cells in response to immunization with OVA-CVC1302-ISA206. LNs were harvested at 14 dpi from mice immunized with OVA-CVC1302-ISA206, lymphocytes were stained with CD4-FITC and I-Ab OVA323-339 tetramer-APC, then OVA⁺ CD4⁺ T cells were adoptively transferred into recipient mice 1 day prior to immunization. FTY720 was administrated intraperitoneally (i.p.) at 0 h and 48 h, and lymph nodes were harvested 72 h later. (A) Mice were immunized in the ear, which was excised 1 h after immunization. Representative confocal images of the localization of transferred CD4⁺ T cell (yellow) to lymph node niches. (B) Mice were immunized in the ear. Representative confocal images of the localization of transferred CD4⁺ T cell (yellow) to lymph node niches.