IL-27 produced during acute malaria infection regulates Plasmodium-specific memory CD4+ T cells.

Malaria infection elicits both protective and pathogenic immune responses, and IL-27 is a critical cytokine that regulate effector responses during infection. Here, we identified a critical window of CD4+ T cell responses that is targeted by IL-27. Neutralization of IL-27 during acute infection with Plasmodium chabaudi expanded specific CD4+ T cells, which were maintained at high levels thereafter. In the chronic phase, Plasmodium-specific CD4+ T cells in IL-27-neutralized mice consisted mainly of CD127+ KLRG1- and CD127- KLRG1+ subpopulations that displayed distinct cytokine production, proliferative capacity, and are maintained in a manner independent of active infection. Single-cell RNA-seq analysis revealed that these CD4+ T cell subsets formed independent clusters that express unique Th1-type genes. These IL-27-neutralized mice exhibited enhanced cellular and humoral immune responses and protection. These findings demonstrate that IL-27, which is produced during the acute phase of malaria infection, inhibits the development of unique Th1 memory precursor CD4+ T cells, suggesting potential implications for the development of vaccines and other strategic interventions.

Analytical approaches for antimalarial antibody responses to confirm historical and recent malaria transmission: an example from the Philippines.

Background: Assessing the status of malaria transmission in endemic areas becomes increasingly challenging as countries approach elimination. Serology can provide robust estimates of malaria transmission intensities, and multiplex serological assays allow for simultaneous assessment of markers of recent and historical malaria exposure. Methods: Here, we evaluated different statistical and machine learning methods for analyzing multiplex malaria-specific antibody response data to classify recent and historical exposure to Plasmodium falciparum and Plasmodium vivax. To assess these methods, we utilized samples from a health-facility based survey (n = 9132) in the Philippines, where we quantified antibody responses against 8 P. falciparum and 6 P. vivax-specific antigens from 3 sites with varying transmission intensity. Findings: Measurements of antibody responses and seroprevalence were consistent with the 3 sites' known endemicity status. Among the models tested, a machine learning (ML) approach (Random Forest model) using 4 serological markers (PfGLURP R2, Etramp5.Ag1, GEXP18, and PfMSP119) gave better predictions for P. falciparum recent infection in Palawan (AUC: 0.9591, CI 0.9497-0.9684) than individual antigen seropositivity. Although the ML approach did not improve P. vivax infection predictions, ML classifications confirmed the absence of recent exposure to P. falciparum and P. vivax in both Occidental Mindoro and Bataan. For predicting historical P. falciparum and P. vivax transmission, seroprevalence and seroconversion rates based on cumulative exposure markers AMA1 and MSP119 showed reliable trends in the 3 sites. Interpretation: Our study emphasizes the utility of serological markers in predicting recent and historical exposure in a sub-national elimination setting, and also highlights the potential use of machine learning models using multiplex antibody responses to improve assessment of the malaria transmission status of countries aiming for elimination. This work also provides baseline antibody data for monitoring risk in malaria-endemic areas in the Philippines. Funding: Newton Fund, Philippine Council for Health Research and Development, UK Medical Research Council.

Induction of liver-resident memory T cells and protection at liver-stage malaria by mRNA-containing lipid nanoparticles.

Recent studies have suggested that CD8+ liver-resident memory T (TRM) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver TRM cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. TRM cells (CD8+ CD44hi CD62Llo CD69+ KLRG1-) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of Plasmodium berghei ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8+ T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease.

mRNA vaccine against malaria tailored for liver-resident memory T cells.

Malaria is caused by Plasmodium species transmitted by Anopheles mosquitoes. Following a mosquito bite, Plasmodium sporozoites migrate from skin to liver, where extensive replication occurs, emerging later as merozoites that can infect red blood cells and cause symptoms of disease. As liver tissue-resident memory T cells (Trm cells) have recently been shown to control liver-stage infections, we embarked on a messenger RNA (mRNA)-based vaccine strategy to induce liver Trm cells to prevent malaria. Although a standard mRNA vaccine was unable to generate liver Trm or protect against challenge with Plasmodium berghei sporozoites in mice, addition of an agonist that recruits T cell help from type I natural killer T cells under mRNA-vaccination conditions resulted in significant generation of liver Trm cells and effective protection. Moreover, whereas previous exposure of mice to blood-stage infection impaired traditional vaccines based on attenuated sporozoites, mRNA vaccination was unaffected, underlining the potential for such a rational mRNA-based strategy in malaria-endemic regions.

Type I interferon production elicits differential CD4+ T-cell responses in mice infected with Plasmodium berghei ANKA and P. chabaudi.

Plasmodium parasites that infect humans are highly polymorphic, and induce various infections ranging from an asymptomatic state to life-threatening diseases. However, how the differences between the parasites affect host immune responses during blood-stage infection remains largely unknown. We investigated the CD4+ T-cell immune responses in mice infected with P. berghei ANKA (PbA) or P. chabaudi chabaudi AS (Pcc) using PbT-II cells, which recognize a common epitope of these parasites. In the acute phase of infection, CD4+ T-cell responses in PbA-infected mice showed a lower involvement of Th1 cells and a lower proportion of Ly6Clo effector CD4+ T cells than those in Pcc-infected mice. Transcriptome analysis of PbT-II cells indicated that type I interferon (IFN)-regulated genes were expressed at higher levels in both Th1- and Tfh-type PbT-II cells from PbA-infected mice than those from Pcc-infected mice. Moreover, IFN-α levels were considerably higher in PbA-infected mice than in Pcc-infected mice. Inhibition of type I IFN signaling increased PbT-II and partially reversed the Th1 over Tfh bias of the PbT-II cells in both PbA- and Pcc-infected mice. In the memory phase, PbT-II cells in PbA-primed mice maintained higher numbers and exhibited a better recall response to the antigen. However, recall responses were not significantly different between the infection groups after re-challenge with PbA, suggesting the effect of the inflammatory environment by the infection. These observations suggest that the differences in Plasmodium-specific CD4+ T-cell responses between PbA- and Pcc-infected mice were associated with the difference in type I IFN production during the early phase of the infection.

CD49d marks Th1 and Tfh-like antigen-specific CD4+ T cells during Plasmodium chabaudi infection.

Upon activation, specific CD4+ T cells up-regulate the expression of CD11a and CD49d, surrogate markers of pathogen-specific CD4+ T cells. However, using T-cell receptor transgenic mice specific for a Plasmodium antigen, termed PbT-II, we found that activated CD4+ T cells develop not only to CD11ahiCD49dhi cells, but also to CD11ahiCD49dlo cells during acute Plasmodium infection. CD49dhi PbT-II cells, localized in the red pulp of spleens, expressed transcription factor T-bet and produced IFN-γ, indicating that they were type 1 helper T (Th1)-type cells. In contrast, CD49dlo PbT-II cells resided in the white pulp/marginal zones and were a heterogeneous population, with approximately half of them expressing CXCR5 and a third expressing Bcl-6, a master regulator of follicular helper T (Tfh) cells. In adoptive transfer experiments, both CD49dhi and CD49dlo PbT-II cells differentiated into CD49dhi Th1-type cells after stimulation with antigen-pulsed dendritic cells, while CD49dhi and CD49dlo phenotypes were generally maintained in mice infected with Plasmodium chabaudi. These results suggest that CD49d is expressed on Th1-type Plasmodium-specific CD4+ T cells, which are localized in the red pulp of the spleen, and can be used as a marker of antigen-specific Th1 CD4+ T cells, rather than that of all pathogen-specific CD4+ T cells.

Plasmodium berghei Hsp90 contains a natural immunogenic I-Ab-restricted antigen common to rodent and human Plasmodium species.

Thorough understanding of the role of CD4 T cells in immunity can be greatly assisted by the study of responses to defined specificities. This requires knowledge of Plasmodium-derived immunogenic epitopes, of which only a few have been identified, especially for the mouse C57BL/6 background. We recently developed a TCR transgenic mouse line, termed PbT-II, that produces CD4+ T cells specific for an MHC class II (I-Ab)-restricted Plasmodium epitope and is responsive to both sporozoites and blood-stage P. berghei. Here, we identify a peptide within the P. berghei heat shock protein 90 as the cognate epitope recognised by PbT-II cells. We show that C57BL/6 mice infected with P. berghei blood-stage induce an endogenous CD4 T cell response specific for this epitope, indicating cells of similar specificity to PbT-II cells are present in the naïve repertoire. Adoptive transfer of in vitro activated TH1-, or particularly TH2-polarised PbT-II cells improved control of P. berghei parasitemia in C57BL/6 mice and drastically reduced the onset of experimental cerebral malaria. Our results identify a versatile, potentially protective MHC-II restricted epitope useful for exploration of CD4 T cell-mediated immunity and vaccination strategies against malaria.

Host-pathogen interaction in the tissue environment during Plasmodium blood-stage infection.

Human malarial infection occurs after an infectious Anopheles mosquito bites. Following the initial liver-stage infection, parasites transform into merozoites, infecting red blood cells (RBCs). Repeated RBC infection then occurs during the blood-stage infection, while patients experience various malarial symptoms. Protective immune responses are elicited by this systemic infection, but excessive responses are sometimes harmful for hosts. As parasites infect only RBCs and their immediate precursors during this stage, direct parasite-host interactions occur primarily in the environment surrounded by endothelial lining of blood vessels. The spleen is the major organ where the immune system encounters infected RBCs, causing immunological responses. Its tissue structure is markedly altered during malarial infection in mice and humans. Plasmodium falciparum parasites inside RBCs express proteins, such as PfEMP-1 and RIFIN, transported to the RBC surfaces in order to evade immunological attack by sequestering themselves in the peripheral vasculature avoiding spleen or by direct immune cell inhibition through inhibitory receptors. Host cell production of regulatory cytokines IL-10 and IL-27 limits excessive immune responses, avoiding tissue damage. The regulation of the protective and inhibitory immune responses through host-parasite interactions allows chronic Plasmodium infection. In this review, we discuss underlying interaction mechanisms relevant for developing effective strategies against malaria.

Activation and IL-10 production of specific CD4+ T cells are regulated by IL-27 during chronic infection with Plasmodium chabaudi.

IL-27, a regulatory cytokine, plays critical roles in the prevention of immunopathology during Plasmodium infection. We examined these roles in the immune responses against Plasmodium chabaudi infection using the Il-27ra-/- mice. While IL-27 was expressed at high levels during the early phase of the infection, enhanced CD4+ T cell function and reduction in parasitemia were observed mainly during the chronic phase in the mutant mice. In mice infected with P. chabaudi and cured with drug, CD4+ T cells in the Il-27ra-/- mice exhibited enhanced CD4+ T-cell responses, indicating the inhibitory role of IL-27 on the protective immune responses. To determine the role of IL-27 in detail, we performed CD4+ T-cell transfer experiments. The Il-27ra-/- and Il27p28-/- mice were first infected with P. chabaudi and then cured using drug treatment. Plasmodium-antigen primed CD4+ T cells were prepared from these mice and transferred into the recipient mice, followed by infection with the heterologous parasite P. berghei ANKA. Il-27ra-/- CD4+ T cells in the infected recipient mice did not produce IL-10, indicating that IL-10 production by primed CD4+ T cells is IL-27 dependent. Il27p28-/- CD4+ T cells that were primed in the absence of IL-27 exhibited enhanced recall responses during the challenge infection with P. berghei ANKA, implying that IL-27 receptor signaling during the primary infection affects recall responses in the long-term via the regulation of the memory CD4+ T cell generation. These features highlighted direct and time-transcending roles of IL-27 in the regulation of immune responses against chronic infection with Plasmodium parasites.

Role of IL-10 in inhibiting protective immune responses against infection with heterologous Plasmodium parasites.

Malaria is induced by infection with Plasmodium parasites, which are genetically diverse, and the immune response to Plasmodium infection has both allele-specific and cross-reactive components. To determine the role of the cross-reactive immune response in the protection and disease manifestation in heterologous Plasmodium infection, we used infection models of P. chabaudi chabaudi (Pcc) and P. berghei ANKA (PbA). CD4+ T cells primed with Pcc infection exhibited strong cross-reactivity to PbA antigens. We infected C57BL/6 mice with Pcc and subsequently treated them with an anti-Plasmodium drug. The Pcc-primed mice exhibited reduced parasitemia and showed no signs of experimental cerebral malaria after infection with PbA. CD4+ T cells from the Pcc-primed mice produced high levels of IFN-γ and IL-10 in response to PbA early after PbA infection. The blockade of IL-10 signaling with anti-IL-10 receptor antibody increased the proportion of activated CD4+ and γδ T cells and the IFN-γ production by CD4+ T cells in response to PbA antigens, while markedly reducing the levels of parasitemia. In contrast, IL-10 blockade did not have a significant effect on parasitemia levels in unprimed mice after PbA infection. These data suggest a potent regulatory role of IL-10 in the cross-reactive memory response to the infection with heterologous Plasmodium parasites leading to the inhibition of the protective immunity and pathogenesis.

CD8+ T Cell Activation Leads to Constitutive Formation of Liver Tissue-Resident Memory T Cells that Seed a Large and Flexible Niche in the Liver.

Liver tissue-resident memory T (Trm) cells migrate throughout the sinusoids and are capable of protecting against malaria sporozoite challenge. To gain an understanding of liver Trm cell development, we examined various conditions for their formation. Although liver Trm cells were found in naive mice, their presence was dictated by antigen specificity and required IL-15. Liver Trm cells also formed after adoptive transfer of in vitro-activated but not naive CD8+ T cells, indicating that activation was essential but that antigen presentation within the liver was not obligatory. These Trm cells patrolled the liver sinusoids with a half-life of 36 days and occupied a large niche that could be added to sequentially without effect on subsequent Trm cell cohorts. Together, our findings indicate that liver Trm cells form as a normal consequence of CD8+ T cell activation during essentially any infection but that inflammatory and antigenic signals preferentially tailor their development.

Defects in centromeric/pericentromeric histone H2A T120 phosphorylation by hBUB1 cause chromosome missegregation producing multinucleated cells.

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.

Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells.

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8+ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4-/- CD8+ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4-mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory-like CD8+ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4-/- CD8+ T cells was impaired, whereas their number eventually reached a level similar to that of wild-type CD8+ T cells. Conversely, memory-like Irf4-/- CD8+ T cells underwent homeostatic proliferation in a manner similar to that of wild-type memory CD8+ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8+ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8+ T cells, whereas the maintenance of memory CD8+ T cells is IRF4-independent.

Zinc finger-IRF composite elements bound by Ikaros/IRF4 complexes function as gene repression in plasma cell.

The transcription factor (TF) interferon regulatory factor-4 (IRF4) promotes both germinal center (GC) reactions and plasma cell (PC) differentiation by binding to alternative DNA motifs including AP-1-IRF composite elements, Ets-IRF composite elements (EICEs), and interferon sequence response elements (ISREs). Although all of these motifs mediate transcriptional activation by IRF4, it is still unknown how some of the IRF4 target genes are downregulated upon PC differentiation. Here, we revealed a molecular mechanism of IRF4-mediated gene downregulation during PC differentiation. By combining IRF4 chromatin immunoprecipitation sequence and gene expression analysis, we identified zinc finger-IRF composite elements (ZICEs) in IRF4 binding regions aligned with genes whose expression was downregulated in PCs. The zinc finger TFs Ikaros and Aiolos were identified as IRF4 binding partners in PCs, and Ikaros but not Aiolos was essential for IRF4 binding to the ZICE sequence and for PC differentiation. The Ebf1 gene, which positively controls B-cell activation and GC reactions, was identified as one of the Ikaros/IRF4 target genes. Importantly, while the ZICE embeds the ISRE motif, IRF4 bound the ZICE motif as heterodimers with Ikaros for repression of target genes, which include Ebf1 In contrast, if the zinc finger motif is juxtaposed to the EICE motif, the Ikaros/PU.1/IRF4 complex functioned to activate target gene expression. Our findings revealed a novel mode of IRF4 activity upon PC differentiation where upon forming an Ikaros/IRF4 DNA-bound complex, a subset of genes is repressed.

Nonspecific CD8+ T Cells and Dendritic Cells/Macrophages Participate in Formation of CD8+ T Cell-Mediated Clusters against Malaria Liver-Stage Infection.

CD8+ T cells are the major effector cells that protect against malaria liver-stage infection, forming clusters around Plasmodium-infected hepatocytes and eliminating parasites after a prolonged interaction with these hepatocytes. We aimed to investigate the roles of specific and nonspecific CD8+ T cells in cluster formation and protective immunity. To this end, we used Plasmodium berghei ANKA expressing ovalbumin as well as CD8+ T cells from transgenic mice expressing a T cell receptor specific for ovalbumin (OT-I) and CD8+ T cells specific for an unrelated antigen, respectively. While antigen-specific CD8+ T cells were essential for cluster formation, both antigen-specific and nonspecific CD8+ T cells joined the clusters. However, nonspecific CD8+ T cells did not significantly contribute to protective immunity. In the livers of infected mice, specific CD8+ T cells expressed high levels of CD25, compatible with a local, activated effector phenotype. In vivo imaging of the liver revealed that specific CD8+ T cells interact with CD11c+ cells around infected hepatocytes. The depletion of CD11c+ cells virtually eliminated the clusters in the liver, leading to a significant decrease in protection. These experiments reveal an essential role of hepatic CD11c+ dendritic cells and presumably macrophages in the formation of CD8+ T cell clusters around Plasmodium-infected hepatocytes. Once cluster formation is triggered by parasite-specific CD8+ T cells, specific and unrelated activated CD8+ T cells join the clusters in a chemokine- and dendritic cell-dependent manner. Nonspecific CD8+ T cells seem to play a limited role in protective immunity against Plasmodium parasites.

Modulation of immune responses by Plasmodium falciparum infection in asymptomatic children living in the endemic region of Mbita, western Kenya.

Individuals living in malaria endemic areas become clinically immune after multiple re-infections over time and remain infected without apparent symptoms. However, it is unclear why a long period is required to gain clinical immunity to malaria, and how such immunity is maintained. Although malaria infection is reported to induce inhibition of immune responses, studies on asymptomatic individuals living in endemic regions of malaria are relatively scarce. We conducted a cross-sectional study of immune responses in asymptomatic school children aged 4-16years living in an area where Plasmodium falciparum and Schistosoma mansoni infections are co-endemic in Kenya. Peripheral blood mononuclear cells were subjected to flow cytometric analysis and cultured to determine proliferative responses and cytokine production. The proportions of cellular subsets in children positive for P. falciparum infection at the level of microscopy were comparable to the negative children, except for a reduction in central memory-phenotype CD8+ T cells and natural killer cells. In functional studies, the production of cytokines by peripheral blood mononuclear cells in response to P. falciparum crude antigens exhibited strong heterogeneity among children. In addition, production of IL-2 in response to anti-CD3 and anti-CD28 monoclonal antibodies was significantly reduced in P. falciparum-positive children as compared to -negative children, suggesting a state of unresponsiveness. These data suggest that the quality of T cell immune responses is heterogeneous among asymptomatic children living in the endemic region of P. falciparum, and that the responses are generally suppressed by active infection with Plasmodium parasites.

Metformin Promotes the Protection of Mice Infected With Plasmodium yoelii Independently of γδ T Cell Expansion.

Adaptive immune responses are critical for protection against infection with Plasmodium parasites. The metabolic state dramatically changes in T cells during activation and the memory phase. Recent findings suggest that metformin, a medication for treating type-II diabetes, enhances T-cell immune responses by modulating lymphocyte metabolism. In this study, we investigated whether metformin could enhance anti-malaria immunity. Mice were infected with Plasmodium yoelii and administered metformin. Levels of parasitemia were reduced in treated mice compared with those in untreated mice, starting at ~2 weeks post-infection. The number of γδ T cells dramatically increased in the spleens of treated mice compared with that in untreated mice during the later phase of infection, while that of αß T cells did not. The proportions of Vγ1+ and Vγ2+ γδ T cells increased, suggesting that activated cells were selectively expanded. However, these γδ T cells expressed inhibitory receptors and had severe defects in cytokine production, suggesting that they were in a state of exhaustion. Metformin was unable to rescue the cells from exhaustion at this stage. Depletion of γδ T cells with antibody treatment did not affect the reduction of parasitemia in metformin-treated mice, suggesting that the effect of metformin on the reduction of parasitemia was independent of γδ T cells.

SIRPα+ dendritic cells regulate homeostasis of fibroblastic reticular cells via TNF receptor ligands in the adult spleen.

In secondary lymphoid organs, development and homeostasis of stromal cells such as podoplanin (Pdpn)-positive fibroblastic reticular cells (FRCs) are regulated by hematopoietic cells, but the cellular and molecular mechanisms of such regulation have remained unclear. Here we show that ablation of either signal regulatory protein α (SIRPα), an Ig superfamily protein, or its ligand CD47 in conventional dendritic cells (cDCs) markedly reduced the number of CD4+ cDCs as well as that of Pdpn+ FRCs and T cells in the adult mouse spleen. Such ablation also impaired the survival of FRCs as well as the production by CD4+ cDCs of tumor necrosis factor receptor (TNFR) ligands, including TNF-α, which was shown to promote the proliferation and survival of Pdpn+ FRCs. CD4+ cDCs thus regulate the steady-state homeostasis of FRCs in the adult spleen via the production of TNFR ligands, with the CD47-SIRPα interaction in cDCs likely being indispensable for such regulation.

Activation and exhaustion of antigen-specific CD8+ T cells occur in different splenic compartments during infection with Plasmodium berghei.

The spleen is the major organ in which T cells are primed during infection with malaria parasites. However, little is known regarding the dynamics of the immune responses and their localization within the splenic tissue during malaria infection. We examined murine CD8+ T cell responses during infection with Plasmodium berghei using recombinant parasites expressing a model antigen ovalbumin (OVA) protein and compared the responses with those elicited by Listeria monocytogenes expressing the same antigen. OVA-specific CD8+ T cells were mainly activated in the white pulp of the spleen during malaria infection, as similarly observed during Listeria infection. However, the fates of these activated CD8+ T cells were distinct. During infection with malaria parasites, activated CD8+ T cells preferentially accumulated in the red pulp and/or marginal zone, where cytokine production of OVA-specific CD8+ T cells decreased, and the expression of multiple inhibitory receptors increased. These cells preferentially underwent apoptosis, suggesting that T cell exhaustion mainly occurred in the red pulp and/or marginal zone. However, during Listeria infection, OVA-specific CD8+ T cells only transiently expressed inhibitory receptors in the white pulp and maintained their ability to produce cytokines and become memory cells. These results highlighted the distinct fates of CD8+ T cells during infection with Plasmodium parasites and Listeria, and suggested that activation and exhaustion of specific CD8+ T cells occurred in distinct spleen compartments during infection with malaria parasites.

Development of a 68Ge/68Ga Generator System Using Polysaccharide Polymers and Its Application in PET Imaging of Tropical Infectious Diseases.

Gallium-68 (68Ga) is a positron emitter for clinical positron emission tomography (PET) applications that can be produced by a 68Ge/68Ga generator without cyclotron. However, commercially available 68Ge/68Ga generator systems require multiple steps for the preparation of 68Ga radiopharmaceuticals and are sometimes plagued by metallic impurities in the 68Ga eluent. We developed a 68Ge/68Ga generator system using polysaccharide-based adsorbents and direct application of the generator-eluted 68Ga-citrate to PET imaging of tropical infectious diseases. N-Methylglucamine (MG) as a 68Ge-adsorbing unit (Sepha-MGs) was introduced to a series of Sephadex G-10, G-15, G-25, G-50, and G-75. In the batch method, over 97% of the 68Ge in the solution was adsorbed onto the Sepha-MG series within 15 min. In particular, 68Ge was effectively adsorbed on the Sepha(15)-MG packed columns and 70-80% of the 68Ga was eluted by 1 mL of 0.1 M trisodium citrate with low 68Ge contamination (<0.001%). The chemical form of the generator-eluted 68Ga solution was identified as 68Ga-citrate. In PET studies, affected regions in mice infected with Leishmania and severe fever with thrombocytopenia syndrome virus were clearly visualized using the 68Ga-citrate. Sepha-MGs are useful adsorbents for 68Ge/68Ga generator systems with high 68Ga elution efficiency and minimal 68Ge breakthrough. These results indicated that eluted 68Ga-citrate can be directly used for PET imaging of infectious sites in mice. This novel generator system may be useful for straightforward PET imaging of infection in clinical practice.