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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 .......

Representative Results

Here, the applied methodology for isolation of CFSE-labeled Plasmodium-infected RBCs in a coculture assay with cytotoxic lymphocytes is described. First, we provide a schematic representation of how to perform the protocol, employing human samples infected with P. vivax (Figure 1). Then, an illustrated flowchart on how to proceed with the protocol in a malaria experimental model using a C57BL/6 mouse infected with P. yoelii (Figure 2)........

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.

Acknowledgements

We thank Dr. Dhelio Pereira and the members of Research Center for Tropical Medicine of Rondônia (CEPEM) for malaria patient enrollment and blood collection and Felicia Ho for helping with manuscript revision. The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium yoelii subsp. yoelii, Strain 17XNL:PyGFP, MRA- 817, contributed by Ana Rodriguez. This research was supported by Lemann Brazil Research Fund, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - 437851/2018-4, fellowships (CJ, GC, CG), and Fundação de Amparo do Estado de Minas Gerais (FAPEMIG) - APQ-00653-16, APQ-02962-1....

Materials

NameCompanyCatalog NumberComments
100 μM cell strainerCorning431752
96 Well Round (U) Bottom Plate Thermo Scientific12-565-65
Anti-human CD235a (Glycophorin A) AntibodyBiolegend349114Used - APC anti-human CD235, dilution 1:100
Anti-human CD3 AntibodyBiolegend317314Used - PB anti-human CD3, dilution 1:200
Anti-human CD8 AntibodyBiolegend344714Used - APC/Cy7 anti-human CD8, dilution 1:200
Anti-human TCR Vδ2 AntibodyBiolegend331408Used - PE anti-human TCR Vδ2, dilution 1:200
Anti-mouse CD8a Antibody Biolegend100733Used- PerCP/Cyanine5.5 anti-mouse CD8a, dilution 1:200
Anti-mouse TER-119/Erythroid Cells AntibodyBiolegend116223Used - APC/Cyanine7 anti-mouse TER-119, dilution 1:200
CellTrace CFSE Cell Proliferation KitInvitrogenC34554
Fetal Bovine Serum, qualifiedGibco26140079
Ficoll-Paque Plus Cytiva17144003Lymphocyte Separation Medium (LSM)
Heparin Sodium Injection, USPmeithel pharma71228-400-003Used - 2000 USP units/2mL
Isoflurane Piramal critical care 66794-0013-25
LS MACS ColumnMiltenyi Biotec130-042-401
LSRFortessa Cell AnalyzerBD Bioscience 
PercollCytiva17089101Density Gradient Separation Medium (DGSM)
QuadroMACS SeparatorMiltenyi Biotec130-090-976
RPMI 1640 MediumGibco11875093
Sodium bicarbonate, powder,  BioReagentSigma-Aldrich  S5761
Syringe With Sub-Q needle - 1mL, 26 gauge; BD14-829-10F
Vacutainer Heparin Tube Glass Green 10 ml BD366480

References

  1. WHO. Global Technical Strategy for Malaria 2016-2030, 2021 Update. World Health Organization. , (2021).
  2. Hafalla, J. C., Silvie, O., Matuschewski, K. Cell biology and immunology of malaria. Immunological Reviews. 240

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