1. The generation of iFMDV-pulsed autologous monocyte-derived DCs
The maturation of DCs is characterized by the upregulated expression of MHC II molecules and costimulatory molecules like CD80, and CD86 33, which determines the capacity of antigen-presentation and T-cell priming 34. To generate MoDCs in vitro, we first differentiated autologous DCs from monocytes isolated from the periphery blood of each pig using porcine GM-CSF and IL-4-containing complete medium, as previously reported 29. The morphological observation showed that typical dendrites on the differentiating cells occurred on days 5 to 7 of culture (Fig. 1A). Flow cytometric analysis showed that around 80% of differentiated cells expressed porcine CD172 and MHC II molecule, consistent with the definition of porcine DCs in previous reports 35. Meanwhile, these cells highly expressed CD1, CD14, CD80, and CD163 molecules (Fig. 1B), indicating the maturation of the differentiated MoDCs 35. In parallel to the most-used inactivated FMDV vaccine, purified FMDV virions were characterized, inactivated, and used as antigens (iFMDV) (Fig.S1). Of note, compared to the mock treatment, iFMDV pulsing upregulated CD163 expression but did not affect the expression of CD1, CD14, CD80, and MHC II (Fig. 1B). However, further LPS treatment after pulsing with iFMDV promoted the maturation of MoDCs as the expressions of CD1, CD14, and CD163 were significantly upregulated (Fig. 1B, 1C, and Fig.S2). These results suggested that iFMDV-pulsed autologous MoDCs were effectively generated and mature.
2. Pig vaccinated with autologous iFMDV-DC induces dominant FMDV-specific IFN-γ-producing CD4 T cells and cytotoxic T lymphocyte responses.
A schematic diagram of autologous DC vaccine generation and experimental design was summarized in Fig. 2. After the preparation of iFMDV-DC, these cells were intramuscularly injected into the original pigs, respectively, and the immune response induced by the iFMDV-DC was examined, and compared to DC alone vaccination, conventional inactivated FMDV vaccine (iFMDV-206) and the control group at different time-points (Fig. 2B). As pulsing DCs with iFMDV represents, classical exogenous antigen presentation 36 and Th1 immunity may be first activated 37. We examined antigen-specific IFN-γ-producing T cells in periphery blood upon ex vivo re-stimulation with or without iFMDV. As shown in Fig. 3A and B, FMDV-specific CD4+ T cells were detected in all four groups at 7 dpv (Fig. 3A and B), and iFMDV-DC immunization induced significantly more FMDV-specific CD4+ T cells than the DC alone, iFMDV-206 and the control group. However, FMDV-specific CD8+ T cells (CD4-CD8α+) were detected only in the iFMDV-DC group but not in other groups (Fig. 3A and B). To confirm the difference in IFN-γ-producing T cells among four groups, total FMDV-specific IFN-γ-producing T cells were detected by a porcine IFN-γ ELISPOT assay. As shown in (Fig. 3C), iFMDV-DC immunization elicited the highest numbers of FMDV-specific IFN-γ-producing T cells than any other group. These results suggested that autologous iFMDV-DC immunization induced predominant FMDV-specific Th1 immune responses in pigs.
While DC-presenting exogenous antigen activates CD4+ T cells, it is also capable of cross-priming CD8+ T cells 19, and several studies have shown that DC vaccine-induced potent cytotoxic CD8+ T cells in humans and mice 19, 23, 24. Therefore, we also examined the cytotoxic T cells induced by autologous iFMDV-DC by flow cytometry using a surrogate marker, CD107a, whose cell surface mobilization indicates the killing activity of NK and CTLs 38, 39. As shown in Fig. 4, upon re-stimulation with iFMDV, all the γδ T, CD4+, CD8+ T cells, and NK (CD3-CD8α+) cells from periphery blood significantly upregulated CD107a expression on the cell surface at 7 dpv, with some background expression (Fig. 3A and B). However, only the iFMDV-DC vaccine-induced statistically highest number of FMDV-specific CD107a-positive CD4+, CD8+ T cells, and NK cells but not γδ T cells, compared to the DC alone, iFMDV-206 and the control group, respectively (Fig. 4A and B). These results implied that the autologous iFMDV-DC vaccine elicits robust FMDV-specific cytotoxic T lymphocyte responses in pigs.
3. iFMDV-DC vaccine induces rapid development of memory CD4 and CD8 T cells and high NAb titers, concomitantly early protection against FMDV in pigs.
DC vaccination has been shown to accelerate the development of memory T cells in mice which mediates early protection against Listeria monocytogenes19. Therefore, we analyzed the changes in memory CD4 and CD8 T cells in pigs after iFMDV-DC vaccination. Swine effector (TEM) and central memory (TCM) CD4 T cells have been defined by surface marker CD27 and CD8α 40, whereas CD8 TEM and TCM cells were defined by CD45RA, CD27, and CCR7 41. CD4 TCM (CD4+CD27+CD8α+) and CD4 TEM (CD4+CD27-CD8α+) were significantly increased in the iFMDV-DC-immunized pigs in terms of percentage at 7 days after immunization (Fig.S3A and Fig. 5). However, there was no significant difference in the CD4 TCM and TEM percentage in the other three groups before and after immunization (Fig. 5B). Similarly, the iFMDV-DC vaccine induced significantly more CD8 early TEM (CD45RA-CCR7-CD27+) and late TEM (CD45RA-CD27-CCR7+) at 7 dpv (Fig. 5D) while the percentage of CD8 TCM (CD45RA-CCR7+CD27+) decreased. No significant difference in the percentage of CD8 early TEM and late TEM was observed in the other three groups before and after immunization.
NAbs play critical roles in the protective immunity to FMDV 42. We also detected the NAb titers among different groups after immunization. Both the iFMDV-DC and iFMDV-206 induced NAbs (1:32–64) and FMDV-specific antibodies (1:22–32) at 7 dpv, compared to the DC and control groups (Fig. 6A and B). However, the iFMDV-DC induced significantly higher NAb titer than iFMDV-206 (Fig. 6A and B).
Considering that the iFMDV-DC elicited rapid development of memory CD4 and CD8 T cells and higher NAb titer shortly after immunization, we expected that the iFMDV-DC vaccine might provide rapid and early protection against FMDV. Therefore, we chose to challenge those pigs with FMDV O/BY/2010 strain at 7 days after immunization and compared the protective efficacy of each group. iFMDV-DC vaccine provided complete protection against the challenge without any clinical signs and viremia throughout the study (Fig. 6C). However, no protection was observed in the other three groups, with viremia peaking at 3 dpc and high clinical scores. These results indicated that the iFMDV-DC vaccine induces rapid development of memory CD4 and CD8 T cells and high NAb titer associated with early protection against FMDV.
4. Autologous iFMDV-DC-induced early protection is associated with rapid secondary T cell response after FMDV challenge.
Upon re-infection, memory T cells have been shown to rapidly differentiate into secondary effector T cells and increase faster than naïve T cells, thus mediating immune protection 43, 44, 45, 46. To further identify the mechanism by which iFMDV-DC induced early protection, we analyzed the secondary response of memory T cells in blood or spleen at 3, 7, and 14 days after FMDV challenge by flow cytometry and porcine IFN-γ ELISPOT assay. We found that there were significantly more numbers of FMDV-specific total IFN-γ-secreting cells (Fig. 7A) and IFN-γ-producing CD4+ and CD8+ T cells (Fig. 7B and C) as well as cytotoxic CD107a+CD8+ T cells (Fig. 7D) in the blood of iFMDV-DC-immunized pigs, compared to the other groups, at 3 and 7 dpc. However, no difference in the percentages of these T cell subsets was observed at 14 dpc among these groups though FMDV-specific T cell responses were detected at this time-point (Fig. 7A, B, C, and D). These results could suggest that iFMDV-DC-induced memory T cells rapidly differentiate into secondary effector T cells early after challenge, whereas primary effector T cells responses in all the four groups caught up to similar levels at 14 dpc in the blood. Further analysis of secondary T cell response in the spleen showed that iFMDV-DC-immunized pigs had significantly more FMDV-specific IFN-γ-secreting cells (Fig. 7E), IFN-γ+CD8+ T cells and CD107a+CD8+ T cells (Fig. 7G and H) but not IFN-γ+CD4+ T cells (Fig. 7F) at 14 dpc, compared to the other groups, suggesting secondary T cells response in the spleen is delayed relative to that in the blood.
To further address whether T cells in the iFMDV-DC-immunized pigs differentiate into effector T cells faster upon challenge, we examined T cell proliferation using Ki67 as a surrogate marker47. Indeed, we observed a significantly higher percentage of Ki67-positive CD4+ and CD8+ T cells but not γδ T cells in the spleens of the iFMDV-DC-immunized pigs (Fig. 8A, B, and S3D), suggesting faster proliferation of these T cell subsets. These results indicated that autologous iFMDV-DC-induced early protection is associated with the rapid proliferation of FMDV-specific secondary effector T cells after the FMDV challenge.
5. Secondary CD8 T EM may contribute more than neutralizing antibodies to the early protection elicited by autologous iFMDV-DC.
Given the importance of NAb in the protective immunity to inactivated FMDV vaccines 42, we further examined the role of NAb in the secondary immune response and early protection elicited by autologous iFMDV-DC after the challenge. As shown in Fig. 9A and B, high titers of FMDV-specific LPB-ELISA antibodies and NAbs were detected in the sera of pigs from the control, iFMDV-206, and DC alone group at 7 dpc, which were significantly increased compared to the NAb titers at 0 dpc (or 7 dpv) (Fig. 6A and B) in these groups. Surprisingly, the titers of LPB-ELISA antibody and NAbs in the sera of the iFMDV-DC-immunized pigs were less increased, barely beyond an indicative threshold of protection (1:64), suggesting that NAbs may play less important roles in the early protection elicited by the iFMDV-DC. To corroborate this finding, we detected and compared the numbers of overall effector memory T cells after the challenge, phenotypically representing cytokine-expressing and cytotoxic CD4+ and CD8+ T cells 48. As shown in Fig. 9C, there was the highest percentage of secondary CD8 TEM but not CD4 TEM in the blood of the iFMDV-DC-immunized pigs, compared to the other three groups at 7 dpc. However, this difference was not observed at 14 dpc (Fig. 9D), and an even lower percentage of CD4 TEM was evident in the iFMDV-DC group (Fig. 9E) compared to the other groups. These results indicated that secondary CD8 TEM may contribute more than NAbs to the early protection elicited by the iFMDV-DC.