Infectious nature of Plasmodium berghei sporozoite modulates APCs to induce better liver-stage memory CD8+T cells

Abstract

Developing effective anti-malarial vaccine has been a challenge. Vaccination with radiation-attenuated sporozoites (RAS) or genetically-attenuated sporozoites (GAP) has shown to confer complete sterile protection against Plasmodia liver-stage (LS) infection that lasts about six to nine months in mice. We have found that the intermittent infectious sporozoite (Inf. Spz) challenge to immune mice following RAS vaccination extends the longevity of sterile protection by maintaining CD8+T cell memory responses to LS infection. In the present study, we observed that a specialized population of dendritic cells (DCs), known for antigen cross-presentation, accumulates in the liver of mice that received Inf. Spz at a frequency that is at least three times higher. Our results are consistent with the others as CD8+DCs increased in number in the livers of mice immunized with RAS or GAP. As the accumulation of CD8+DCs in the liver was thought to be a result of migration of the same from circulation, the expression of chemokines (Ccl-20 and Ccl-21) in the liver of mice was measured. The prominent expression of chemokines in liver of mice inoculated with Inf. Spz. suggestive of the fact that greater accumulation of CD8+DCs at the site of infection is because of the infectious status of the sporozoite. Further, the capacity of DCs to modulate T cells is predominantly dependent on their activation status, which involves the up-regulation of major-histocompatibility (MHCs) and elevated expression of costimulatory molecules (CD80, CD86, and CD40) on the surface of DCs. Therefore, we studied the role of infectious status of sporozoites in modulating CD8+DCs. Our results, in fact, suggest that expression level of MHCs and co-stimulatory molecules significantly induced by Inf. Spz meet the requirements for better T cell activation and memory formation. Similarly, CD40-CD40L signalling is known to be involved in cross presentation and APC licensing to promote survival of antigen specific CD8+T cells. Therefore, Inf. Spz inoculation/challenge that enhances not only the accumulation of CD8+DCs in the liver, but also favored generation of higher frequencies of activated CD4+T cells with greater expression of CD40L. This in turn might promote APC licensing of CD8+DCs and provide help to CD8+T cells. The expression of CD40 or CD40L on CD8+T cells promoted by Inf. Spz challenge provides us a plausible explanation that CD8+T cells, either receive signals from the licensed CD8DCs through CD40-CD40L or from CD4+T cells through CD40L-CD40 or from both to become a qualitatively better LS memory CD8+T cells Subsequently, it is known that microenvironment at the site of infection plays a critical role in determining the state of APCs. Therefore, we did attempt to understand whether microenvironment in liver created following Inf. Spz inoculation could be influenced by the infectious status of sporozoite. The level of chemokine expression, and modulation TLR signals promoting type-I interferon expression was favored by the Inf. Spz inoculation or challenge. The type-I IFN, major inflammatory cytokines during initiation of immune response are critical in bridging innate and adaptive immune response. The key functions of type-I IFN response involve activation and differentiation of DCs by promoting the expression of MHC molecules and various co-stimulatory molecules for the priming of T cells. Furthermore, type-I interferons promote the cross-presentation of antigen to the CD8+T cells and are known to favor formation of memory CD8+T cells. In contrast, the MHC class-II antigen processing and presentation pathway related gene expression was upregulated in liver of mice following Inf. Spz inoculation, but not in RAS inoculated mouse liver. This showed that MHC class-II processing and presentation pathway was activated in the liver of Inf. Spz inoculated mice. Our data generated from the present study supports the idea that the infectious status of sporozoite promotes MHC-II mediated response, a critical requirement for APCs to be licensed. Thus, CD4+T cell activation could be induced following Inf. Spz inoculation. Our results suggest that the Inf. Spz challenge to prior RAS immunized mice modulates the CD8+DCs, which might be shaping the fate of memory CD8+T cells against Plasmodium LS infection. Whole genome RNA-sequencing (RNA-seq.) analysis supports the idea that the infectious status of sporozoite promotes MHC-II mediated antigen processing and presentation response, a critical requirement for APCs to be licensed. This study provides clues to the nature of innate immune responses that a protective malaria vaccine would require. Further, the production of type-I interferon and induced signaling appear to shape the protective CD8+T cell response. In conclusion, our study might help in elucidating the immunological determinants for generation of long-lived protective immunity.