We thank Dr. Dhelio Pereira and the members of Research Center for Tropical Medicine of Rond&#244;nia (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 Cienti&#769;fico e Tecnolo&#769;gico (CNPq) - 437851/2018-4, fellowships (CJ, GC, CG), and Funda&#231;&#227;o de Amparo do Estado de Minas Gerais (FAPEMIG) - APQ-00653-16, APQ-02962-18; Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) - fellowship (LL).

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 Rond&#244;nia (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 of the Brazilian Practice Guide for the Care and Use of Animals for Scientific and Didactic Purposes of the National Council for the Control of Animal Experimentation (CONCEA). The protocols were approved by the Fiocruz Animal Experimentation Council (CEUA protocol LW15/20-2).
1. Collection of human blood samples and PBMC isolation
<ol>
	<li>Collect blood of Plasmodium-infected patients in a 10 mL evacuated blood collection tube with sodium heparin. As malaria patients display lymphopenia, there is a range of 5-9 x 106 peripheral blood mononuclear cells (PBMCs) in a 10 mL blood sample. Since CD8+ T cells or &#947;&#948; T cells make up ~10% of PBMCs, preferably collect 50-100 mL of blood/patient. 
	NOTE: A minimum of 1 x 105 effector cells/ condition should be considered.</li>
	<li>Calculate the percentage of iRBCs by blood smear as described below.
	<ol>
		<li>Add 5 &#956;L of total blood to a clear slide and prepare a blood smear. Perform Romanowsky-type panoptic fast stain or May-Gu&#776;nwald-Giemsa stain. Here, the panoptic fast stain kit is used, which is composed of three reagents: reagent A (fixation), reagent B (cytoplasmic stain), and reagent C (nuclear and cytoplasmic differential stain).</li>
		<li>Slowly dip the slide in solution A 10x, then 4x in solution B, and finally 10x in solution C. Drain out any excess reagent from the slides between solutions. After dipping in solution C, rinse the slide in running tap water and let it dry.</li>
		<li>Under an upright light microscope with a 100x oil immersion objective, count 1000 RBCs in sequential squares and calculate the percentage of parasitemia using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/63987/63987eq01.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Dilute 15 mL of blood in a 1:1 proportion in sterile phosphate-buffered saline (PBS).</li>
	<li>Add 15 mL of lymphocyte separation medium (density of 1.077g/mL) to a 50 mL tube. Carefully layer the 30 mL of diluted blood sample onto the centrifugation medium solution. When layering the sample, do not let the blood sample and lymphocyte separation medium mix.</li>
	<li>Centrifuge the tubes at 400 x g for 40 min at 22 &#176;C, with low acceleration and no break setting.</li>
	<li>Draw off the upper layer containing plasma using a sterile pipette, leaving the mononuclear cell layer undisturbed. Transfer the layer of mononuclear cells (PBMCs) to a sterile tube using a sterile pipette.</li>
	<li>Do not discard the tube containing the blood pellet as it will be used later for infected RBC isolation. From this step, be sure to keep the RBCs at room temperature (RT). Never let them cool down.</li>
	<li>Wash the cells twice by adding PBS and centrifuge at 350 x g for 10 min, with break setting. Resuspend the cell pellet in 5 mL of RPMI medium supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS; complete medium).</li>
	<li>Count the PBMCs in the presence of trypan blue solution to check cell viability, using a hemacytometer (Neubauer chamber) or automated cell counter. Do not count the blue-stained cells as these represent dying cells, which absorb trypan blue.</li>
	<li>Adjust the cell concentration to 107 cells/mL using complete medium. Purify the desired cytotoxic lymphocyte populations (CD8+ T cells, NK cells, &#947;&#948; T cells) using magnetic beads isolation as per reagent manufacturer protocol.</li>
</ol>
2. Human RBC isolation
NOTE: For human-infected RBC isolation, it is recommended to start with blood samples that have a minimum of 2% parasitemia, preferentially with more in the trophozoite/early schizont parasite stage.
<ol>
	<li>Prepare the PERCOLL (from hereafter referred to as density gradient separation medium) at the recommended concentration as described below.
	<ol>
		<li>Add 90 mL of 100% density gradient separation medium and 10 mL of 10x PBS to obtain 90% isotonic density gradient separation medium.</li>
		<li>For P. vivax-infected reticulocyte separation, prepare45% density gradient separation media. Add 50 mL of 90% isotonic density gradient separation medium and 50 mL of 1x PBS to obtain 45% density gradient separation medium.</li>
		<li>For P. falciparum-infected erythrocyte separation, prepare 65% density gradient separation media. Add 72 mL of 90% isotonic density gradient separation medium and 28 mL of 1x PBS to obtain 65% density gradient separation medium.</li>
		<li>For uninfected reticulocyte separation, prepare 70% density gradient separation media. Add 78 mL of 90% isotonic density gradient separation medium and 22 mL of 1x PBS to obtain 70% density gradient separation medium.</li>
	</ol>
	</li>
	<li>Warm the density gradient separation medium to 37 &#176;C in a water bath.</li>
	<li>After removal of the PBMC layer, carefully remove as much of the top layer of centrifugation medium as possible without touching the cells. With a glass Pasteur pipette, carefully remove the upper neutrophil layer without disturbing the RBC pellet and estimate pellet volume. Add 4x the RBC pellet volume of RT complete medium and resuspend.</li>
	<li>In a second 50 mL conical tube, add 5x the pellet volume of 45%, 65%, or 70% density gradient separation medium depending on the cell type to isolate. Layer the RBC suspension on top of the density gradient separation medium layer carefully.</li>
	<li>Spin for 15 min at 850 x g, with low acceleration and no break setting. Collect the cloudy red/brown layer lying in between the supernatant and density gradient separation medium with a 5 mL pipette. Transfer to a sterile 15 mL tube.</li>
	<li>Wash the cells by adding RT complete medium up to 15 mL and spin down for 10 min at 860 x g. Repeat the washing by adding 10 mL of RT complete medium and spin down. Discard the supernatant and prepare a blood smear from the pellet to check for iRBC enrichment, following step 1.2.</li>
	<li>Resuspend the pellet in 1 mL of RT complete medium and let sit at room temperature while counting the cells. Add 10 &#956;L of cell suspension in a hemocytometer (Neubauer chamber) and count the RBCs in the central area subdivided into 25 medium squares. Adjust cell concentration to 1 x 107 iRBCs/mL in RT complete media.</li>
</ol>
3. Experimental malaria infection in mouse
<ol>
	<li>Thaw an aliquot of cryopreserved P. yoelii 17XNL:PyGFP (MRA-817), a GFP-expressing strain obtained from MR4/ATCC, and inject 100 &#956;L intraperitoneally (i.p.) in an 8-week-old female C57BL/6 mouse.</li>
	<li>Follow the parasitemia burden every 3 days by tail vein lancing blood collection.
	<ol>
		<li>Puncture the vessel with the needle bevel up, entering the vein at a shallow angle beginning at the distal end of the tail.</li>
		<li>Collect the blood sample with a pipette or capillary tube up to 5 or 10 mL, then apply manual pressure to stop the bleeding.</li>
		<li>Prepare a blood smear (as described in step 1.2) until parasitemia reaches 10%-15% of infected RBCs.</li>
	</ol>
	</li>
	<li>Collect 10 &#956;L of blood by tail vein lancing and dilute to 100 &#956;L of PBS to i.p. inject into the second donor mouse. 
	NOTE: Cryopreserved parasites should be thawed and passed in mice twice before being used for experimental infections.</li>
	<li>When the second passage reaches 15% of parasitemia, collect five drops of blood by the tail clipping method and dilute in 1 mL of PBS. Prepare an infection solution by adjusting the concentration to 1 x 106 iRBCs/mL in sterile PBS and inject i.p. 100 &#956;L (1 x 105 iRBCs) of the solution into each mouse required for the experiment.</li>
	<li>Monitor parasitemia every 2-3 days until it reaches ~30% iRBCs, which occurs approximately 12 days post-infection. When the mice reach the desired parasitemia, collect blood by cardiac puncture as described below.
	<ol>
		<li>Aspirate 100 &#181;L of heparin solution (30 U/mL) into a 1 mL syringe with a 26 G needle.</li>
		<li>Anesthetize the mouse by inhalation with 5% isoflurane and confirm the absence of reflexes. Place the mouse on its side and perpendicularly insert the needle just below the elbow, through the ribs, and into the heart. Pull out the syringe plunger slowly and rotate the needle until 0.5-1 mL of blood is obtained.</li>
		<li>Perform humane euthanasia by cervical dislocation under anesthesia. Aseptically clean the left side of the mouse with 70% ethanol. With surgical scissors, make a cut on the left side of the mouse passing through the skin and peritoneum. Locate the spleen and remove it.</li>
		<li>Place the mouse spleen in a Petri dish with 5 mL of complete medium to purify the desired effector cell population (e.g., CD8+ T cells).</li>
	</ol>
	</li>
</ol>
4. Obtaining fresh whole mouse splenocytes
<ol>
	<li>In the Petri dish, carefully cut the spleen into small pieces using scissors or a razor.</li>
	<li>Place a 100 &#181;M cell strainer over a 50 mL conical tube and transfer the excised spleen into the cell strainer with a pipette. Mash the spleen through the strainer with a syringe plunger.</li>
	<li>Wash the cells through the strainer with 10 mL of complete media. Centrifuge the cells at 300 x g for 10 min at 4 &#176;C, then discard the supernatant.</li>
	<li>Resuspend the cell pellet in 2 mL of cold 1x RBC lysis buffer. Incubate the suspension for 5 min on ice.</li>
	<li>Wash the cell suspension with 10 mL of 4 &#176;C complete media. Repeat the wash step three times and remove any cell clots between washes for spleens from Plasmodium-infected mice.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at 4 &#176;C, then discard the supernatant. 
	NOTE: Plasmodium-infected spleens are enlarged and filled with phagocytic cells containing degraded hemoglobin and hemozoin.</li>
	<li>To avoid any issues during the magnetic bead isolation of effector cells, use the following steps to remove hemozoin-enriched phagocytic cells and hemozoin. Resuspend the splenic cells with MACS buffer and adjust concentration to 1 x 108 cells/mL. Place an LS or LD column in the magnetic field. Prepare the column by rinsing with 3 mL of MACS buffer.</li>
	<li>Apply cell suspension onto the column. Wash the column three times with 3 mL of MACS buffer. Collect flow-through containing the splenic cells.</li>
	<li>Count the splenocytes in a trypan blue solution and check cell viability in a hemacytometer (Neubauer chamber) or automated cell counter. Adjust cell concentration to 1 x 107 cells/mL in RT complete media.</li>
</ol>
5. Cytotoxic effector cell purification (CD8a+ T cell negative selection)
NOTE: There are many positive and negative selection reagents that can purify cytotoxic effector cells (CD8+ T, &#947;&#948; T, NK, iNKT, MAIT cells). In this protocol, we use a negative selection of splenic CD8a+ T cells and follow manufacturer instructions.
<ol>
	<li>Centrifuge all splenocytes at 400 x g for 10 min at 4 &#176;C and discard the supernatant. Resuspend the cell pellet in 40 &#181;L of MACS buffer.</li>
	<li>Add 10 &#181;L of a biotin-antibody cocktail. Mix well and incubate for 5 min on ice.</li>
	<li>Add 30 &#181;L of MACS buffer. Add 20 &#181;L of anti-biotin micro-beads. Mix well and incubate for 10 min on ice.</li>
	<li>Add 400 &#181;L of MACS buffer and proceed to magnetic cell separation. Place the MACS LS column in the magnetic field support. Prepare the column by rinsing with 3 mL of MACS buffer.</li>
	<li>Apply cell suspension onto the column. Wash the column three times with 3 mL of MACS buffer. Collect the flow-through containing all unlabeled cells, which are the enriched CD8a+ T cells.</li>
	<li>Count the CD8a+ T cells in a trypan blue solution and check cell viability in a hemacytometer (Neubauer chamber) or automated cell counter. Adjust the cell concentration to 1 x 107 cells/mL in RT complete media.</li>
</ol>
6. P. yoelii infected RBC isolation
<ol>
	<li>Centrifuge the collected blood in a 1.5 mL tube at 850 x g for 3 min. Discard the serum and resuspend the blood in 1 mL of 1x RPMI without FBS.</li>
	<li>Place the LS column in the magnetic field support and rinse with 3 mL of 1x RPMI. Pass the RBC suspension through the column. To isolate more iRBCs, reapply the flowthrough (3 mL of RPMI and 1 mL of diluted blood) into the column.</li>
	<li>Wash twice with 5 mL of 1x RPMI. Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. Do not let any columns dry up.</li>
	<li>Add 5 mL of RPMI, remove the column, and purge the iRBCs into a new 15 mL tube. Count the iRBCs and adjust the concentration to 1 x 107 RBCs/mL.</li>
</ol>
7. CFSE labeling of RBCs and preparation for flow cytometry
NOTE: Start the RBC labeling protocol with twice the number of cells that will be used for the experiment, since ~50% of the cells are typically lost in the washing steps following the CFSE labeling step. To establish the autofluorescence of the RBCs/iRBCs, include a control sample of unlabeled cells.
<ol>
	<li>Dilute CFSE to a final concentration of 10 mM in 1x RPMI without FBS. Wash the cells once with 1x RPMI without FBS in a 15 mL tube.</li>
	<li>Resuspend the RBCs in 500 &#956;L of 1x RPMI without FBS and add 500 &#956;L of the diluted CFSE. Incubate for 8 min at RT, protected from light.</li>
	<li>Wash three times by adding 14 mL of complete medium (10% FBS RPMI), followed by centrifugation at 850 x g for 10 min. Resuspend the cells in complete medium to a concentration of 1-5 x 106/ mL. Incubate for 1 h at RT.</li>
</ol>
8. Cytotoxic lymphocyte/RBC coculture and preparation for flow cytometry
NOTE: If the involvement of specific receptors or molecules will be measured, incubate the specific blocking and isotype control antibodies (10 mg/mL) with the effector cells for 30 min before coculture.
<ol>
	<li>Plate the cells in a 96-well round-bottom plate. Add the purified lymphocytes and CFSE-labeled iRBCs in the desired effector-target cell ratio to a final volume of 200 &#956;L and homogenize. Prepare each condition in triplicate. 
	NOTE: We suggest adjusting the iRBC concentration to 1-5 x 104 cells/mL and choosing the ratio based on the purified cell number (e.g., 0.5:1, 2.5:1, and 5:1).</li>
	<li>Include the spontaneous lysis control, which is the target iRBCs without the effector, to evaluate any spontaneous lysis, as this will be used to represent a 100% cell viability condition.</li>
	<li>Spin down the plate for 1 min at 360 x g to maximize cell contact. Incubate at 37 &#176;C and 5% CO2&#160;for 4 h. 
	NOTE: Hypoxia conditions (low O2), systematically used for P. falciparum culture, should not be used since the effector cells do not survive in this environment. In contrast, Plasmodium parasites are viable in ambient air in 5% CO2&#160;for up to 12 h.</li>
	<li>Spin for 5 min at 850 x g and flip the plate to remove the supernatant. 
	NOTE: If desired, the supernatant can be used to measure any soluble factors released in the coculture.</li>
	<li>Label the RBCs with anti-mouse Ter119 1:200 (or anti-human CD235 1:100 for human samples) and CD8+ T cells with anti-CD8 1:200 or anti-CD3 1:200 antibodies (anti-mouse or human) for 30 min at 4 &#176;C in 1x PBS containing 3% FBS (FACS buffer). 
	NOTE: The antibody fluorophore should be selected based on the cell tracer/cell proliferation reagent (e.g., CFSE-labelled iRBC, APC-Cy7 anti-Ter119, and PerCP-Cy5.5 anti-CD8a).</li>
	<li>Wash the cells with FACS buffer and spin down for 5 min at 850 x g. Transfer the samples to FACS tubes and add 30 &#956;L of counting beads &#65279;to individual tubes. Homogenize the count beads by vortexing for 30 s.</li>
</ol>
9. Flow cytometry
<ol>
	<li>Analyze samples using a 405/488/561/640 laser instrument.</li>
	<li>For human cells labeled with CFSE (FITC), PE anti-human &#947;&#948; TCR, and PE-Cy7 anti-human CD235a antibody, use 530/30 (FITC), 575/25 (PE), and 780/60 (PE-Cy7) filters in a three-laser (Blue, Red, Yellow-Green) configuration cytometer.</li>
	<li>For mouse cells labeled with CFSE (FITC), PerCP-Cy5.5 anti-mouse CD8a, and APC-Cy7 anti-mouse Ter119, use 530/30 (FITC), 695/40 (PerCP-Cy5.5), and 780/60 (APC-Cy7) filters in the three-laser cytometer setup.</li>
	<li>Choose the counting beads population as the stopping gate to acquire a minimum of 20,000 events in the stop gate, which should be identical for all conditions. Perform analysis and compensation using appropriate flow cytometry acquisition/analysis software.</li>
	<li>Set the gating strategy as described below.
	<ol>
		<li>Select single cells (singlets), excluding debris using FSC peak height (H) to area (A) ratio. Select RBCs and exclude the lymphocytes from analysis based on fluorescence of the RBC marker (Ter119 or CD235a) and the lymphocyte-specific antibody (CD8a).</li>
		<li>In the previous RBC gating, select viable RBCs based on CFSE positive&#160;staining. Analyze the data with flow cytometer data analysis software.</li>
	</ol>
	</li>
</ol>
10. Calculation and statistics
<ol>
	<li>To calculate the percentage of iRBC lysis, follow the gating strategy described in step 9. The percentage of viable RBCs is the frequency of CFSE-positive cells inside the RBC gate based on Ter119 (mouse) or CD235a (human) positive fluorescence.</li>
	<li>Use the spontaneous lysis control, RBCs without effector cells, condition as a control to estimate RBC spontaneous rupture. This condition will be considered RBC lysis (100% viability). Use the following formula to calculate the percentage of RBC lysis in each tested condition: 
	<img alt="Equation 2" src="/files/ftp_upload/63987/63987eq02v4.jpg" style="margin: auto;" /> 
	NOTE: During the culture incubation, some infected RBCs may spontaneously lyse or be ruptured by the parasite. Use the CFSE-positive RBC frequency of the spontaneous lysis condition, which contains no effector cells, as the baseline for lymphocyte cytotoxicity.</li>
	<li>Calculate the average of triplicates for each condition and determine statistical significance using two-way ANOVA at multiple comparisons.</li>
</ol>

<ol><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Technical+Strategy+for+Malaria+2016-2030%2C+2021+Update%5BTitle%5D)+AND+%22World+Health+Organization%22%5BJournal%5D)">Global Technical Strategy for Malaria 2016-2030, 2021 Update.</a> World Health Organization. , (2021).</li>
<li>Hafalla, J. C., Silvie, O., Matuschewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+biology+and+immunology+of+malaria%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">Cell biology and immunology of malaria.</a> Immunological Reviews. 240 (1), 297-316 (2011).</li>
<li>Belnoue, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vaccination+with+live+Plasmodium+yoelii+blood+stage+parasites+under+chloroquine+cover+induces+cross-stage+immunity+against+malaria+liver+stage%5BTitle%5D)+AND+%22Journal+of+Immunology%22%5BJournal%5D)">Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage.</a> Journal of Immunology. 181 (12), 8552-8558 (2008).</li>
<li>Leong, Y. W., Lee, E. Q. H., Rénia, L., Malleret, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+malaria+erythrocyte+preference+assessment+by+an+ex+vivo+tropism+assay%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Rodent malaria erythrocyte preference assessment by an ex vivo tropism assay.</a> Frontiers in Cellular and Infection Microbiology. 11, 680136(2021).</li>
<li>Ladeia-Andrade, S., Ferreira, M. U., De Carvalho, M. E., Curado, I., Coura, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-dependent+acquisition+of+protective+immunity+to+malaria+in+riverine+populations+of+the+amazon+basin+of+Brazil%5BTitle%5D)+AND+%22The+American+Journal+of+Tropical+Medicine+and+Hygiene%22%5BJournal%5D)">Age-dependent acquisition of protective immunity to malaria in riverine populations of the amazon basin of Brazil.</a> The American Journal of Tropical Medicine and Hygiene. 80 (3), 452-459 (2009).</li>
<li>Antonelli, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunology+of+Plasmodium+vivax+malaria%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">The immunology of Plasmodium vivax malaria.</a> Immunological Reviews. 293 (1), 163-189 (2020).</li>
<li>Kazmin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systems+analysis+of+protective+immune+responses+to+RTS%2C+S+malaria+vaccination+in+humans%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Systems analysis of protective immune responses to RTS, S malaria vaccination in humans.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (9), 2425-2430 (2017).</li>
<li>Epstein, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+attenuated+malaria+vaccine+designed+to+protect+through+hepatic+CD8%2B+T+cell+immunity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Live attenuated malaria vaccine designed to protect through hepatic CD8+ T cell immunity.</a> Science. 334 (6055), 475-480 (2011).</li>
<li>Draper, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Malaria+vaccines%3A+recent+advances+and+new+horizons%5BTitle%5D)+AND+%22Cell+Host+%26+Microbe%22%5BJournal%5D)">Malaria vaccines: recent advances and new horizons.</a> Cell Host & Microbe. 24 (1), 43-56 (2018).</li>
<li>Junqueira, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%CE%B3%CE%B4+T+cells+suppress+Plasmodium+falciparum+blood-stage+infection+by+direct+killing+and+phagocytosis%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">γδ T cells suppress Plasmodium falciparum blood-stage infection by direct killing and phagocytosis.</a> Nature Immunology. 22 (3), 347-357 (2021).</li>
<li>Junqueira, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+CD8%2B+T+cells+recognize+and+kill+Plasmodium+vivax-infected+reticulocytes%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Cytotoxic CD8+ T cells recognize and kill Plasmodium vivax-infected reticulocytes.</a> Nature Medicine. 24 (9), 1330-1336 (2018).</li>
<li>Arora, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NK+cells+inhibit+Plasmodium+falciparum+growth+in+red+blood+cells+via+antibody-dependent+cellular+cytotoxicity%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity.</a> eLife. 7, 36806(2018).</li>
<li>Paul, F., Roath, S., Melville, D., Warhurst, D. C., Osisanya, J. O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+malaria-infected+erythrocytes+from+whole+blood%3A+use+of+a+selective+high-gradient+magnetic+separation+technique%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Separation of malaria-infected erythrocytes from whole blood: use of a selective high-gradient magnetic separation technique.</a> Lancet. 2 (8237), 70-71 (1981).</li>
<li>Shaw-Saliba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insights+into+an+optimization+of+Plasmodium+vivax+Sal-1+in+vitro+culture%3A+the+aotus+primate+model%5BTitle%5D)+AND+%22PLOS+Neglected+Tropical+Diseases%22%5BJournal%5D)">Insights into an optimization of Plasmodium vivax Sal-1 in vitro culture: the aotus primate model.</a> PLOS Neglected Tropical Diseases. 10 (7), 0004870(2016).</li>
<li>Mehlotra, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+in+vitro+culture+of+Plasmodium+vivax+isolates+from+Madagascar+maintained+in+Saimiri+boliviensis+blood%5BTitle%5D)+AND+%22Malaria+Journal%22%5BJournal%5D)">Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.</a> Malaria Journal. 16 (1), 442(2017).</li>
<li>Hojo-Souza, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+IFN-%CE%B3+and+granulysin+to+the+clearance+of+Plasmodium+yoelii+blood+stage%5BTitle%5D)+AND+%22PLOS+Pathogens%22%5BJournal%5D)">Contributions of IFN-γ and granulysin to the clearance of Plasmodium yoelii blood stage.</a> PLOS Pathogens. 16 (9), 1008840(2020).</li>
<li>Parish, C. R., Glidden, M. H., Quah, B. J. C., Warren, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation%5BTitle%5D)+AND+%22Current+Protocols+in+Immunology%22%5BJournal%5D)">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current Protocols in Immunology. 84 (1), 4-9 (2009).</li>
<li>Migliaccio, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erythroblast+enucleation%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Erythroblast enucleation.</a> Haematologica. 95 (12), 1985(2010).</li>
<li>Grimberg, B. T., Erickson, J. J., Sramkoski, R. M., Jacobberger, J. W., Zimmerman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+Plasmodium+falciparum+growth+and+development+by+UV+flow+cytometry+using+an+optimized+Hoechst-thiazole+orange+staining+strategy%5BTitle%5D)+AND+%22Cytometry+Part+A%3AThe+Journal+of+the+International+Society+for+Analytical+Cytology%22%5BJournal%5D)">Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.</a> Cytometry Part A:The Journal of the International Society for Analytical Cytology. 73 (6), 546-554 (2008).</li>
<li>Pattanapanyasat, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+of+malaria+parasites+in+two+different+red+blood+cell+populations+using+biotin+and+flow+cytometry%5BTitle%5D)+AND+%22Cytometry%3AThe+Journal+of+the+International+Society+for+Analytical+Cytology%22%5BJournal%5D)">Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.</a> Cytometry:The Journal of the International Society for Analytical Cytology. 25 (3), 287-294 (1996).</li>
<li>Bianco, A. E., Battye, F. L., Brown, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasmodium+falciparum%3A+rapid+quantification+of+parasitemia+in+fixed+malaria+cultures+by+flow+cytometry%5BTitle%5D)+AND+%22Experimental+Parasitology%22%5BJournal%5D)">Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry.</a> Experimental Parasitology. 62 (2), 275-282 (1986).</li>
<li>Jacobberger, J. W., Horan, P. K., Hare, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+malaria+parasite-infected+blood+by+flow+cytometry%5BTitle%5D)+AND+%22Cytometry%3AThe+Journal+of+the+International+Society+for+Analytical+Cytology%22%5BJournal%5D)">Analysis of malaria parasite-infected blood by flow cytometry.</a> Cytometry:The Journal of the International Society for Analytical Cytology. 4 (3), 228-237 (1983).</li>
<li>Bei, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+flow+cytometry-based+assay+for+measuring+invasion+of+red+blood+cells+by+Plasmodium+falciparum%5BTitle%5D)+AND+%22American+Journal+of+Hematology%22%5BJournal%5D)">A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.</a> American Journal of Hematology. 85 (4), 234(2010).</li>
<li>Montes, M., Jaensson, E. A., Orozco, A. F., Lewis, D. E., Corry, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+general+method+for+bead-enhanced+quantitation+by+flow+cytometry%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">A general method for bead-enhanced quantitation by flow cytometry.</a> Journal of Immunological Methods. 317 (1-2), 45-55 (2006).</li>
</ol>

The authors declare no competing interests.

Here we describe an in vitro assay to measure Plasmodium-infected red blood cell killing by cytotoxic lymphocytes. This assay &#65279;can help elucidate the mechanisms of cellular protective immunity to the malaria parasite&#39;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.
Importantly, this method can be used to study P. vivax and other Plasmodium parasites that are not maintained in vitro culture14,15. In addition, this protocol can be adapted to any Plasmodium species and different hosts, such as humans, mice, and non-human primates. Flow cytometry-based methods are widely used to evaluate cytotoxic cell activation, cytokine production, and degranulation10,11,16, but the current method is the only one that explores RBC lysis by direct cell contact with killer cells.
There are also many methods, such as microscopy, polymerase chain reaction (PCR), and flow cytometry, that are used to study malaria by measuring parasitemia, invasion, and antimalarial activity. However, to analyze the cell-mediated cytotoxicity of RBCs, current methods that use Cr51&#160;or hemoglobin release are not sensitive enough to evaluate RBC lysis. LDH release may also be used to evaluate RBC killing by some CTLs like CD8+ T cells, but measuring LDH is not an accurate method as activated innate or innate-like cells may kill each other as a regulatory mechanism.
CFSE is widely used to track cells or explore cell proliferation in flow cytometry or fluorescent microscopy17. Since RBCs do not proliferate18, the CFSE fluorescence intensity should decrease with cell lysis. Flow cytometry is a very accurate technique as it can detect very low levels of specific markers, and so it has been widely used to track parasitemia and invasion in malaria infection19,20,21,22,23. Most importantly, flow cytometry can measure many parameters in a single cell and sort out cells into different populations. By adding quantitative standard reagents (counting beads) to each sample, one can normalize the number of acquired events and obtain absolute cell counts24. This method allows for a reliable quantitative comparison between experimental conditions, which is critical for the currently proposed protocol.
Some critical steps for this protocol include ensuring the sample quality, ensuring proper cell staining, and including controls to normalize calculations. A large amount of fresh blood samples is required to purify infected RBCs. The density gradient separation medium purification should be approached carefully as its concentration is critical to obtaining the desired Plasmodium iRBCs. The blood overlay onto the density gradient separation medium must also be done as slowly as possible, and the ring red-layer harvesting must be done carefully to avoid losing iRBCs. To make the purification process easier, a high parasitemia blood sample should be used as the amount of iRBCs will be higher. It should be noted that in the RBC isolation protocol using magnetic fields, only parasite mature stages (middle-late stage trophozoites or schizonts) that produce significant amounts of hemozoin will be purified. As a result, any ring stage will not be collected, and this may affect the final result.
Cell staining should be performed carefully to avoid loss or lysis of cells. Prolonged storage or fixation should be avoided before staining. It is important to note that the experimental conditions should be performed in triplicate using serial effector:target ratios and appropriate controls, such as an unlabeled, spontaneous lysis condition and a hypothesis control. The addition of cell counting beads is crucial as it provides an easy way to determine the concentration of cells or absolute cell counts for each sample. During flow cytometer acquisition, be sure to set the number of events to be collected in the counting beads gate to normalize the number of cells in each sample after coculture.
Since at least 1 x 105 effector cells are needed for consistent results in a coculture assay, one possible limitation of the described methodology is the limited number of cells after purification, which can be a concern especially when working with rare cell populations. Indeed, this applies to iRBC purification, as it requires having a very high parasitemia that is not so easily obtained, especially when working with human samples in endemic areas.
One protocol improvement may be the coculture incubation time. Although we previously used a 12 h incubation time10,11, we recently observed that 4 h is enough to differentiate the experimental conditions, thus reducing the time for potential spontaneous lysis and improving the reproducibility of the results.
The mechanisms involved in the direct killing of Plasmodium-infected RBCs are gradually being uncovered. Our group was the first to show that CD8+ T cells can recognize and kill P.vivax-infected reticulocytes via HLA-I presentation by the infected cell11. Recently, another study demonstrated that &#947;&#948; T cells recognize P. falciparum-infected RBCs through phosphoantigen recognition by butyrophylin, a molecule present on the surface of iRBCs10. In both studies, RBC lysis is granulysin and granzyme B dependent, and leads to intracellular parasite killing.
Although many methods have been developed over the years to measure parasitemia, invasion, antimalarial activity, and image cell interaction, none have been capable of analyzing the direct killing of iRBCs by cytotoxic cells in a quantitative flow cytometry-based assay19,20,21. We used an innovative approach to evaluate cell lysis capacity in malaria by CFSE labeling of Plasmodium-infected RBCs and evaluation of iRBC lysis in a specific period of coculture with lymphocytes. Therefore, this in vitro killing assay presents a novel strategy to clarify the mechanisms of cell-mediated immunity to blood-stage malaria, which will help advance the study of new therapeutic targets and the development of malaria vaccines.

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,&#160;the liver or pre-erythrocytic stage is asymptomatic, and symptoms only occur during the parasite&#39;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 the RBC, the parasites differentiate into trophozoites and schizonts by schizogony, until schizonts rupture the erythrocyte, releasing newly formed merozoites, repeating this blood cycle. &#65279;Repeated cycles of invasion, replication, and merozoite release result in an exponential growth of the parasite population and ultimately trigger disease symptoms2.
An important challenge in studying the immune response to malaria is that the Plasmodium spp. that infects humans does not infect laboratory animal models. Thus, Plasmodium-infected patient samples must be collected fresh and immediately processed and analyzed. However, in malaria-endemic areas, resources to access immunological and molecular mechanisms are limited. Due to these limitations, rodents are widely used as experimental models to investigate the immune response against Plasmodium infection. While P. berghei and P. chabaudi are often used as surrogates for P. falciparum infection, the non-lethal strain of P. yoelii&#160;17XNL also has many features in common with P. vivax, such as reticulocyte restricted infection3,4. The development of Plasmodium in vitro assays, that can be used for human or animal model-derived samples, is valuable in gaining a better understanding of the pathogenesis of malaria and comparing the immunological response elicited by different species of the parasite.
Protective antimalarial immunity is not completely understood either at the pre-erythrocytic stage nor the blood stage. &#65279;It is known that exposure to repeated infections results in partial acquired immunity, but sterile immunity is rarely developed5. &#65279;For decades, anti-Plasmodium protective immunity was mainly associated with the induction of neutralizing or opsonizing antibodies that prevent parasite invasion of host cells or lead to phagocytosis by antigen-presenting cells, respectively6. As a result, most efforts to produce antimalarial vaccines thus far have relied on inducing protective and long-lasting antibodies7,8. However, the sterile protection induced by vaccination with an attenuated sporozoite directly correlates with the activation and expansion of cytotoxic T lymphocytes8,9.
Recently, some studies of freshly isolated patient samples and in vitro cultures have demonstrated that innate or adaptative cytotoxic immune cells like CD8+ T10, &#947;&#948; T11, and NK cells12&#160;can directly eliminate Plasmodium-infected RBCs and its intracellular parasite in an effector:target ratio manner. These seminal findings defined an entirely new immune effector mechanism in the context of malaria. To dissect this novel antimalarial immunity, it is essential to explore cytotoxic effector mechanisms of killer cells against infected-RBCs (iRBCs) in natural infection or vaccination.
Here we present an in vitro assay that measures the cytotoxic activity of lymphocytes against malaria in the blood stage. This assay can then help elucidate the mechanisms of the cellular immune response against the Plasmodium erythrocyte stage. The target cells, iRBCs, are labeled with carboxyfluorescein succinimidyl ester (CFSE) to evaluate cell viability, and are then cocultured with effector cells like cytotoxic lymphocytes (CTL). This coculture is then evaluated by flow cytometry, using fluorescent markers for specific cell types. Finally, the percentage of iRBC lysis by CTL is calculated by dividing the experimental condition by the spontaneous rupture of RBCs and spontaneous lysis control, which occurs during incubation without the effector cell. Overall, this killing assay methodology can contribute to a better understanding of cell-mediated malaria immunity.

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

in vitro assay of plasmodium-infected red blood cell killing by cytotoxic lymphocytes

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 (&#947;&#948; 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, &#947;&#948; 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.

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). In Figure 3, the expected results (before and after) for step 6 of the protocol (enrichment of P. yoelii-infected RBCs using magnetic columns) are shown. Finally, Figure 4&#160;shows a representative flow cytometry analysis, detailing the gating strategy needed to assess the percentage of RBC lysis, as described in step 9. Therefore, this section highlights the different techniques used here, describing the entire process of acquisition, assembly, and data analysis to assess iRBC killing by cytotoxic lymphocytes.
Human Plasmodium-infected RBCs lysis by cytotoxic cells 
A schematic representation of the protocol to evaluate the killing of human Plasmodium-infected RBCs by cytotoxic cells is shown in Figure 1. To measure cell lysis, PBMC from Plasmodium-infected patients was purified using the separation medium, followed by magnetic purification of the cytotoxic lymphocyte (CTL) population of interest (e.g., CD8+ T, &#947;&#948; T, NK, iNKT, MAIT cells, etc.). The RBCs&#39; pellet remaining from the PBMC isolation was used to enrich Plasmodium-infected RBCs using density gradient separation medium (65% P. falciparum; 45% P. vivax). iRBCs were labeled with CFSE and incubated with or without lymphocytes for 4 h at 37 &#176;C, 5% CO2. After the coculture period, all RBCs (infected or not) were labeled with anti-human CD235a antibody (for the RBCs) and CTL-specific antibodies (e.g., anti-CD8, anti- &#947;&#948;TCR, etc.) before flow cytometry analysis. The acquisition and analysis of human samples were similar to those demonstrated for mouse samples in Figure 4.
P. yoelii-infected RBC lysis by cytotoxic CD8+ T cells 
A flow chart of the experimental procedure to evaluate the cytotoxic effector mechanism in the experimental malaria model is shown in Figure 2, along with representative experimental data shown in Figure 3 and Figure 4. C57BL/6 mice were infected with 1 x 105&#160;P. yoelii (Py) iRBCs, and parasitemia was monitored until it reached around 30%. Py-iRBCs were isolated from blood using magnetic separation, as&#160;the parasite heme subproduct, hemozoin, is iron-enriched and functions as paramagnetic particles in a magnetic field13. The purity of the enriched sample was analyzed by blood smear in comparison with the pre-column sample (Figure 3). iRBCs were then labeled with the cell tracer reagent CFSE. In parallel, cytotoxic CD8+ T cells were purified from splenocytes using a magnetic negative selection kit. As a control, the same protocol was used for CD8+ T cell purification from uninfected mice. Effector (CD8+ T) and target (CFSE-labelled Py-iRBC) cells were incubated at different effector:target ratios (E:T: 0.5:1, 2.5:1, and 5:1) for 4 h at 37 &#176;C 5% CO2. At the end of the coculture, each condition was labeled with antibodies anti-mouse Ter119 for RBCs and anti-CD8a for cytotoxic cells. The gating strategy and sample results are represented in Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63987/63987fig01.jpg" /> 
Figure 1. Schematic of the in vitro killing assay. Example of killing assay experiment. Plasmodium-infected blood is collected and processed using a separation gradient medium. After centrifugation, the PBMC layer is collected, and cytotoxic cells (CTLs) were purified using magnetic beads. The RBC layer is once again processed using density gradient separation media. After centrifugation, the Plasmodium-iRBC layer is collected and labeled with CFSE. Thereafter, the purified CTL and iRBCs were cocultured for 4 h at 37 &#176;C, 5% CO2, followed by cell-specific labeling and analysis in a flow cytometer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63987/63987fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63987/63987fig02.jpg" /> 
Figure 2. Experimental malaria model for killing assay. First, C57BL/6 mice were infected with 105 PyX17NL-infected red blood cells. Then, parasitemia is monitored by blood smear, until the peak of infection (30%-40% iRBCs), approximately 12 days post-infection (dpi). On the next day, mice were bled and euthanized for spleen collection. The blood was processed, and infected RBCs were enriched by magnetic separation, followed by labeling with CFSE (upper right). The spleen was processed for cytotoxic lymphocytes isolation, by negative selection using magnetic beads. Afterward, the purified cytotoxic lymphocytes and iRBCs were cocultured for 4 h at 37 &#176;C, 5% CO2, followed by cell-specific labeling and analysis in a flow cytometer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63987/63987fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63987/63987fig03.jpg" /> 
Figure 3. iRBC enrichment using magnetic columns. The iRBCs were purified from P. yoelii infected mice using LS columns. The purification is highlighted by blood smears from blood collected before (A) and after (B) column enrichment. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63987/63987fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63987/63987fig04.jpg" /> 
Figure 4. Assessment of cell lysis percentage of P. yoelii-iRBCs by CD8+ lymphocytes. The gating strategy for cell lysis percentage. (A) Set the stopping gate into the counting beads population (~20,000 events). (B) Control spontaneous lysis, iRBCs without lymphocytes. Start the gating strategy by selecting the single cells, excluding debris, based on the FSC peak height to area ratio, followed by the selection of the RBC population in APC-Cy7 Ter119 positive and PerCP-Cy5.5 CD8&#160;negative. Then select live iRBCs as CFSE (FITC) high positive. (C) Example experiment analysis using different effector: target ratios, displaying the percentage of live RBC after the coculture.&#160;(D) Graphical representation of the RBC lysis percentage calculated based on the formula described in section 10. The significance comparison between the groups (cytotoxic CD8 or na&#239;ve CD8) was assessed by two-way ANOVA with multiple comparisons. P &#60; 0.0001 (****). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63987/63987fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes at JoVE.com

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

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.&#160;

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

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

2.0K Views.  Fundação Oswaldo Cruz. Deze testmethodologie is een belangrijke vooruitgang in de kennis van celimmuniteit tegen malaria in het bloedstadium, helpt nieuwe therapeutische doelen te ontdekken en de voortgang van malariavaccin te versnellen. Het opzetten van een plasmodium in vitro assay die kan worden gebruikt voor menselijke of dierlijke monsters is waardevol voor een beter begrip van malaria pathogenese. Beschermende malaria-immuniteit wordt niet volledig begrepen.Voor de ontwikkeling van nieuwe therapieën ...

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Here, we provide a detailed methodology to isolate and assess cytotoxic function of natural killer cells from placentas by a colorimetric plate assay. The reduced uterine perfusion pressure rat model of placental ischemia was chosen to demonstrate the antibody-mediated isolation and assessment of the cytotoxic function of natural killer cells.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

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

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