CD4+ and CD8+ T cell- and IL-17-mediated protection against Entamoeba histolytica induced by a recombinant vaccine

Abstract

Amebiasis in the murine model can be prevented by vaccination with the Gal/GalNAc lectin through a T cell-dependent mechanism. In this work we further decipher the mechanism of this protection. Mice vaccinated with the recombinant "LecA" fragment of the Gal/GalNAc lectin with alum were capable of transferring protection to naïve recipients by both CD4+ T cells and surprisingly CD8+ T cells. We then examined the cytokine profile of these cells. CD4+ T cells from PBMC of LecA-alum vaccinated mice were observed to be a major source of IFN-γ, known to be a protective cytokine with this vaccine. In contrast, CD8+ T cells produced relatively little IFN-γ but more IL-17 than the CD4 compartment. We thus examined the role of IL-17 in vaccine mediated protection and found through neutralization experiments that this cytokine contributed to LecA-alum vaccine protection. In addition we examined whether these cells exhibited direct amebicidal activity in vitro and found that both populations had amebicidal activity at high concentrations (1000:1) but CD8+ T cells appeared more potent, capable of cytotoxicity at a 100:1 ratio. In conclusion, both CD4 and CD8 T cells exert protection with this amebiasis vaccine. The mechanism of CD8 T cell-mediated protection may include direct amebicidal activity and/or IL-17 production. Both IL-17 and IFN-γ are useful surrogates for immune protection.

Figure 1. LecA/alum-mediated protection is adoptively transferred by CD3+, CD4+ or CD8+ T cells, not serum antibodies

LecA/alum-immunized and PBS/alum sham-immunized CBA donor mice were sacrificed 4 weeks after the final boost. Splenocytes were isolated and enriched for CD3+, CD4+ and CD8+ T cells. (A) The purity of enriched cells was determined by FACS staining for CD3, B220, CD4 and CD8 prior and after enrichment. (B) T cells or serum from vaccinated vs. control donor mice were transferred to naïve recipients, which were challenged with E. histolytica trophozoites 3 days after transfer. The recipients were sacrificed 12 days post-challenge to assess protection as determined by culture of cecal contents. Close bars: recipients that were transferred with cells or serum from LecA-immunized donors; Open bars: recipients that were transferred with cells or serum from PBS control donors. “No transfer” refers to LecA/alum immunized (close bar) or PBS control mice (open bar) directly challenged with E. histolytica without performing adoptive transfer.

Figure 2. IFN-γ- and IL-17-producing CD4 and CD8 T cells in peripheral blood

PBMC were isolated from LecA/alum-immunized and PBS/alum control CBA mice 3 weeks after the final boost. Cells were in vitro stimulated with LecA for 20 hrs and stained for CD4, CD8 and intracellular IFN-γ and IL-17. Cells were gated on IFN-γ+ (A, left) or IL-17+ (B, left) populations for their CD4 or CD8 phenotype (A, B, middle and right, n=16). *** p <0.001.

Figure 3. IL-17 contributes to LecA/alum-mediated protection

Mice immunized with LecA (closed bars) or PBS (open bars) in the alum regimen were administered anti-IL-17 or control IgG from 1 day prior to 4 days after challenge (3 doses total). Mice were sacrificed seven days post-infection. *p <0.05.

Figure 4. In vitro amebicidal activity of immune CD8 and CD4 T cells

LecA/alum-immunized and PBS/alum sham-immunized CBA mice were sacrificed 4 weeks after the final boost. Splenocytes were isolated from vaccinated mice (close circles) or PBS controls (open circles) and enriched for CD8+ (A) and CD4+ (B) T cells. T cells were co-cultured with HM1 E. histolytica trophozoites at various ratios indicated in the×axis. Viability of HM1 co-cultured with (C) CD8+ T cells and (D) CD4+ T cells was determined by trypan blue exclusion. * p <0.05, *** p <0.001.