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 ...

<ol>
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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.
<p class="jove_conte...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#956;M cell strainer</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Round (U) Bottom Plate&#160;</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&#948;2 Antibody</td>
			<td>Biolegend</td>
			<td>331408</td>
			<td>Used - PE anti-human TCR V&#948;2, dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a Antibody&#160;</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&#160;</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&#160;</td>
			<td>Piramal critical care&#160;</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&#160;</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,&#160; BioReagent</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#160;S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe With Sub-Q needle - 1mL, 26 gauge;&#160;</td>
			<td>BD</td>
			<td>14-829-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacutainer Heparin Tube Glass Green 10 ml&#160;</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)....

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 ...

This assay methodology, it's a major advancement in the knowledge of cell immunity against blood stage malaria, helping to uncover new therapeutic targets and to accelerate the progress of malaria vaccine. The establishment of a plasmodium in vitro assay that can be used for human or animal samples is valuable for a better understand of malaria pathogenesis. Protective malaria immunity is not completely understood.For the development of new therapies, it is essential to explore mechanisms of cell immunity to the malaria parasitic stage. Demonstrating the procedure will be me, Luna de Lacerda, and Christopher Gomes, an undergrad student from Caroline's lab. To begin, aspirate 100 microliters of heparin solution into a one-milliliter syringe with a 26-gauge needle.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 to one milliliter of blood is obtained. 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. Place the mouse spleen in a Petri dish with five milliliters of complete medium to purify the desired effector cell population.Place a 100-micromolar cell strainer over a 50-milliliter conical tube and transfer the excised spleen into the cell strainer using anatomical forceps. Mash the spleen through the strainer with a syringe plunger. Wash the cells through the strainer with 10 milliliters of complete media.Centrifuge the cells at 300 G for 10 minutes at four degrees centigrade. Then, discard the supernatant. Resuspend the cell pellet in two milliliters of cold RBC lysis buffer and incubate the suspension for five minutes on ice.Wash the cell suspension with 10 milliliters of four-degrees-centigrade complete media and remove any cell clots between washes for spleens from plasmodium-infected mice. Centrifuge the cells at 400 G for five minutes at four degrees centigrade. Then, discard the supernatant.Now place an LS or LD column in the magnetic field and prepare the column by rinsing it with three milliliters of MACS buffer. Next, apply cell suspension onto the column. After washing the column three times with three milliliters of MACS buffer, collect flow-through containing the splenic cells.Harvest 10 microliters of the splenic cells and add 10 microliters of trypan blue to check cell viability. Add the cells to the hemocytometer. Count the splenocytes in a trypan blue solution and check cell viability.Centrifuge all splenocytes and discard the supernatant. Resuspend the cell pellet in 40 microliters of MACS buffer. Add 10 microliters of a biotin antibody cocktail to the cells, mix well, and incubate for five minutes on ice.After incubating the anti-biotin microbeads on ice for 10 minutes, place the MACS LS column in the magnetic field support and prepare the column by rinsing it with three milliliters of MACS buffer. Apply cell suspension onto the column. Wash the column three times with three milliliters of MACS buffer and collect the flow-through containing all unlabeled cytotoxic cells.After centrifuging the collected blood at 350 times G for five minutes, discard the serum and resuspend the blood in one milliliter of RPMI without FBS. After placing the LS column in the magnetic field support, rinse with RPMI and pass the RBC suspension through the column. Once the cells pass through the column, centrifuge the flow-through, discard the supernatant, and reapply one milliliter of the flow-through into the column to isolate more iRBCs.Wash the column twice with five milliliters of RPMI. Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. Add five milliliters of RPMI, remove the column, and purge the iRBCs into a new 15-milliliter tube.Dilute one microliter of a 10-millimolar CFSE solution in one milliliter RPMI without FBS. After resuspending the RBCs in 500 milliliters of RPMI without FBS, add 500 milliliters of the diluted CFSE and incubate for eight minutes at room temperature, protected from light. Wash the cells thrice with 14 milliliters of complete medium and centrifuge the cells twice.For cytotoxic lymphocyte co-culture, 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 milliliters and homogenize. Prepare each condition in triplicate and incubate the cells at 37 degrees centigrade.The iRBCs were purified from P.yoelii-infected mice using LS columns. The purification is highlighted by blood smears from blood collected before and after column enrichment. The gating strategy needed to assess the percentage of RBC lysis is presented here.The stopping gate is set into the counting beads population. The gating strategy is started by selecting the single cells, excluding debris, based on the forward scatter peak height-to-area ratio, followed by the selection of the RBC population in Ter119+and CD8-Then, select live IBCs as CFSE high positive. Here is the analysis applied to an experimental example using different effectors to target ratios, displaying live RBCs after the co-culture.A graphical representation of the RBC lysis percentage is shown, calculated based on this formula. The significance between the groups using cytotoxic CD8 or naive CD8 was assessed by two-way ANOVA with multiple comparisons. The current method explores RBC lysis by direct cell contact with killer cells, and it can be planned to uncover the involved mechanisms using blocking antibodies to specific receptors or molecules.This in vitro kill assay presents a novel strategy to clarify the mechanisms of cell-mediated immunity to blood stage malaria, which will help the progress of new malarial therapies and vaccines.

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Research

JoVE Journal

Immunology and Infection

1.8K visualizações.  Fundação Oswaldo Cruz. Esta metodologia de ensaio, é um grande avanço no conhecimento da imunidade celular contra a malária em estágio sanguíneo, ajudando a descobrir novos alvos terapêuticos e a acelerar o progresso da vacina contra a malária. O estabelecimento de um ensaio in vitro de plasmódio que possa ser usado para amostras humanas ou animais é valioso para uma melhor compreensão da patogênese da malária. A imunidade protetora contra a malária não é completamente compreendida.Para o dese...