In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Summary

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes. 

Abstract

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8⁺ and gamma delta T cells (γδ T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8⁺ T, γδ T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

Introduction

Malaria remains a global health crisis, with more than 240 million cases and 627,000 malaria-related deaths reported in 2020¹. There are currently five parasitic species that can cause malaria in humans, out of which Plasmodium falciparum and Plasmodium vivax are the two most prevalent species. During Plasmodium infection, the liver or pre-erythrocytic stage is asymptomatic, and symptoms only occur during the parasite's asexual cycle in the erythrocytic stage. At this infection stage, thousands of merozoites derived from the liver stage are released into the bloodstream and infect red blood cells (RBCs). In t....

Protocol

All procedures were conducted following the policies of the Oswaldo Cruz Foundation and the National Ethical Council (CAAE: 59902816.7.0000.5091). The human protocols were developed in collaboration with the clinical research group from the Research Center for Tropical Medicine of Rondônia (CEPEM), which was in charge of enrolling patients in the study. Informed consent was obtained from all the patients.

For the animal study, procedures were performed following the principles of conduct .......

Discussion

Here we describe an in vitro assay to measure Plasmodium-infected red blood cell killing by cytotoxic lymphocytes. This assay can help elucidate the mechanisms of cellular protective immunity to the malaria parasite's erythrocytic stage. The major advantage of this methodology is that it provides a quantitative assay of the cell-mediated killing of iRBCs that can be used to address many questions about how immune cells interact with different Plasmodium spp.

Materials

Name	Company	Catalog Number	Comments
100 μM cell strainer	Corning	431752
96 Well Round (U) Bottom Plate	Thermo Scientific	12-565-65
Anti-human CD235a (Glycophorin A) Antibody	Biolegend	349114	Used - APC anti-human CD235, dilution 1:100
Anti-human CD3 Antibody	Biolegend	317314	Used - PB anti-human CD3, dilution 1:200
Anti-human CD8 Antibody	Biolegend	344714	Used - APC/Cy7 anti-human CD8, dilution 1:200
Anti-human TCR Vδ2 Antibody	Biolegend	331408	Used - PE anti-human TCR Vδ2, dilution 1:200
Anti-mouse CD8a Antibody	Biolegend	100733	Used- PerCP/Cyanine5.5 anti-mouse CD8a, dilution 1:200
Anti-mouse TER-119/Erythroid Cells Antibody	Biolegend	116223	Used - APC/Cyanine7 anti-mouse TER-119, dilution 1:200
CellTrace CFSE Cell Proliferation Kit	Invitrogen	C34554
Fetal Bovine Serum, qualified	Gibco	26140079
Ficoll-Paque Plus	Cytiva	17144003	Lymphocyte Separation Medium (LSM)
Heparin Sodium Injection, USP	meithel pharma	71228-400-003	Used - 2000 USP units/2mL
Isoflurane	Piramal critical care	66794-0013-25
LS MACS Column	Miltenyi Biotec	130-042-401
LSRFortessa Cell Analyzer	BD Bioscience
Percoll	Cytiva	17089101	Density Gradient Separation Medium (DGSM)
QuadroMACS Separator	Miltenyi Biotec	130-090-976
RPMI 1640 Medium	Gibco	11875093
Sodium bicarbonate, powder,  BioReagent	Sigma-Aldrich	S5761
Syringe With Sub-Q needle - 1mL, 26 gauge;	BD	14-829-10F
Vacutainer Heparin Tube Glass Green 10 ml	BD	366480