3.1 Conventional dendritic cells show enhanced costimulatory property and MHC expression along with an increased population in the anionic (L3) adjuvant treated group
CD11c+CD8a+ cDC played major role in antigen presentation [34], [35]. The population percentage of cDC gated out from the splenocytes (Fig 1A) has no significant difference between N3 and L3 groups (Fig 1B). Even in their lowest doses (L3III and N3III), both the adjuvants are capable of inducing an enhanced cDC population than the non-adjuvant group (Fig 1B). Apart from the percentages, the cDC population is also been represented here in absolute number for better comparison (Fig 1C). When we studied the costimulatory marker CD80 and CD86 expression, we find the percentage of cDC having double-positive for these two markers is significantly higher in N3 treated groups as compared to the L3 groups (Fig 1D), especially in higher doses of antigen (N3I). The same goes true for MHCI and MHCII double-positive cDC population percentages (Fig 1D). The analysis of the mean fluorescence intensity however showed the higher expression of MHCI in the N3 groups, while MHCII is more abundant in the L3 groups (Fig 1E). The expression of DEC205, a cross-presenting molecule on the cDC surface [36] however shows that the L3 group has the highest expression (Fig 1F).
These findings on cDC are significant because they showed a differential response in its part after receiving differentially charged adjuvants. Enhanced double positive cDC with CD80/86, MHCI/ II expression in N3 treatment, and comparatively higher expression of MHCII and DEC205 in L3 treatment, provides us the clue for differential modulation of humoral or cell-mediated immunity downstream.
3.2 Cytotoxic CD8T cells and its costimulatory marker CD28 expression in the L3 groups
CD3+ cells are gated out and the population percentage of CD3+CD4+ and CD3+CD8+T cells have been studied (Fig 2A). Cytotoxic CD3+CD8+ T cells are indispensable for cell-mediated immunity. The population percentage of the CD8T cells was higher in the N3 adjuvant treatments, even in the lower dose of antigens (Fig 2B). The absolute number of CD3+CD8+ T cells is represented for better comparison (Fig: 2C). The MFI calculated for the CD28 expression on the cells stained with CD3+CD8+CD28+ gives a stronger signal even in the N3III group (Fig 2D).
Significantly higher CD8T cell population with enhanced CD28 expression along with the higher MHCI expression in the cDC of the N3 groups (as compared to the L3 treated groups) we can conclude an undergoing upregulation of cell-mediated immunity post cationic adjuvant treatment.
3.3 Higher population percentage of CD4T cell with the altered skewing of Treg: Th17 ratio observed in adjuvant treated groups
The population percentage of the CD4T cells showed non-significant changes in the anionic L3 group and the anionic N3 group, although both the groups showed a significant increase of the same in comparison to non-adjuvant control at high doses (L3I and N3I) (Fig 3A). The absolute number of CD3+CD4+ T cells is represented for better comparison (Fig: 3B). An interesting finding comes out when we studied the expression of RAR-related orphan receptor gamma (RORγT) and forkhead box P3 (FOXP3) on these CD3+CD4+ T cells. Regulatory T cells express FOXP3, while more aggressive Th17 cells express RORγT [37]. CD3+CD4+FOXP3+RORγT- Treg cell population is much higher in the anionic L3 groups especially with the lower dose of antigen (L3II and L3III) (Fig 3C). On contrary, CD3+CD4+FOXP3-RORγT+ Th17 cells are of higher percentage in the cationic N3 group (Fig 3D). Even the lowest dose N3III showed a significantly higher Th17 cell population as compared to non-adjuvant control (Fig 3D).
The Treg cell population is more tolerogenic, while Th17 shows a more aggressive response and drives the Th1 type of immune response [31], [37] The imbalance in Treg/Th17 has been reported in multiple diseases before [37]–[39]. A low Treg: Th17 ratio in anionic N3 groups is observed as compared to the L3 groups (Fig 3E) indicating aggressive Th1 response in cationic treated groups.
Our findings on the CD4T cells are significant as we can conclude that despite sharing almost similar CD4T population percentage in the differentially charged adjuvant groups, cationic N3 groups having more Th17 subsets than Tregs, are more aggressive in eliminating the pathogens.
3.4 N3 treatment induces significantly higher interferon-gamma (IFNγ) producing T cell subsets
The CD3+CD4+ and CD3+CD8+ T cell subsets gated out as shown previously in Fig 2A. The permeabilized cellular subsets with IFNγ staining showed significantly higher IFNγ producing CD4T cells and CD8T cells in N3 treatment groups than with L3 treatments (Fig 4A). This data along with the previous findings of enhanced T cell immunity via Enzyme-linked immunosorbent spot (ELISPOT) assay post N3 treatment [19] indicates a dependency of cell-mediated immunity in the cationic adjuvant treatment. Both this and the previous ELISPOT reports [19] that CD8T is the main effector cell in the N3 groups while the cell-mediated immunity is based more on CD4T cells in the L3 treatment.
3.5 Less immature B cell and more Plasma B cells in L3 group indicates elevated B cell response in anionic adjuvant treatment
B220+CD19+CD27- immature B cells [33]showed a higher population in the N3 groups as compared to L3 groups (Fig 4B,4C). The plasma B cells (B220lowCD19-CD27highCD38low) population is significantly higher in the L3 groups even in the lower doses as compared to the N3 groups as well as with the non-adjuvant control (Fig 4C). No significant increase of the Plasma B cells is observed in the N3 groups as compared with the control.
Significantly higher activated plasma B cells and less immature B cells in the L3 treatment as compared to N3 treatment indicates an enhanced humoral immunity post anionic L3 adjuvant treatment.
3.6 Serum IgG level against influenza HA shows significantly high expression in both L3I and N3I groups, With higher virus neutralization capacity in N3I group
To assess the humoral response provided by these two adjuvants, we have performed ELISA to investigate the IgG and IgA antibody titer specific for the HA antigen of influenza. As shown in Fig 4A, both the adjuvants express a significantly higher titer of anti-HA IgG as compared to the non-adjuvant control of the same antigen dose. While comparing between the two adjuvant groups, L3 groups (L3I and L3II) express significantly higher antibody expression than the N3 groups even at the highest antigenic dose (Fig 5A). It is also to be noted that anti-HA IgA responses are critically low in the N3 treated groups (Fig 5B). Only L3 treatment can induce anti-HA IgA which is significantly higher in both the N3 treated and non-adjuvant groups.
While studying the neutralization capacity of these serums against the influenza virus we observe a significant neutralizing capacity in both the L3I and N3I groups, with the L3I group exhibiting a much stronger response than the N3 groups (Fig 5C).
Thus, we conclude L3 treatment exhibit significantly higher humoral response with more immunoglobulin synthesis and neutralization potential as compared with the N3 groups.