T cells expressing multiple co-inhibitory molecules in acute malaria are not exhausted but exert a suppressive function in mice.

Overwhelming activation of T cells in acute malaria is associated with severe outcomes. Thus, counter-regulation by anti-inflammatory mechanisms is indispensable for an optimal resolution of disease. Using Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice, we performed a comprehensive analysis of co-inhibitory molecules expressed on CD4+ and CD8+ T cells using an unbiased cluster analysis approach. We identified similar T cell clusters co-expressing several co-inhibitory molecules like programmed cell death protein 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) in the CD4+ and the CD8+ T cell compartment. Interestingly, despite expressing co-inhibitory molecules, which are associated with T cell exhaustion in chronic settings, these T cells were more functional compared to activated T cells that were negative for co-inhibitory molecules. However, T cells expressing high levels of PD-1 and LAG-3 also conferred suppressive capacity and thus resembled type I regulatory T cells. To our knowledge, this is the first description of malaria-induced CD8+ T cells with suppressive capacity. Importantly, we found an induction of T cells with a similar co-inhibitory rich phenotype in Plasmodium falciparum-infected patients. In conclusion, we demonstrate that malaria-induced T cells expressing co-inhibitory molecules are not exhausted, but acquire additional suppressive capacity, which might represent an immune regulatory pathway to prevent further activation of T cells during acute malaria.

Deciphering the Plasmodium falciparum malaria-specific CD4+ T-cell response: ex vivo detection of high frequencies of PD-1+TIGIT+ EXP1-specific CD4+ T cells using a novel HLA-DR11-restricted MHC class II tetramer.

Relatively little is known about the ex vivo frequency and phenotype of the Plasmodium falciparum-specific CD4+ T-cell response in humans. The exported protein 1 (EXP1) is expressed by plasmodia at both, the liver stage and blood stage, of infection making it a potential target for CD4+ and CD8+ effector T cells. Here, a fluorochrome-labelled HLA-DRB1∗11:01-restriced MHC class II tetramer derived from the P. falciparum EXP1 (aa62-74) was established for ex vivo tetramer analysis and magnetic bead enrichment in 10 patients with acute malaria. EXP1-specific CD4+ T cells were detectable in 9 out of 10 (90%) malaria patients expressing the HLA-DRB1∗11 molecule with an average ex vivo frequency of 0.11% (0-0.22%) of total CD4+ T cells. The phenotype of EXP1-specific CD4+ T cells was further assessed using co-staining with activation (CD38, HLA-DR, CD26), differentiation (CD45RO, CCR7, KLRG1, CD127), senescence (CD57), and co-inhibitory (PD-1, TIGIT, LAG-3, TIM-3) markers as well as the ectonucleotidases CD39 and CD73. EXP1-specific tetramer+ CD4+ T cells had a distinct phenotype compared to bulk CD4+ T cells and displayed a highly activated effector memory phenotype with elevated levels of co-inhibitory receptors and activation markers: EXP1-specific CD4+ T cells universally expressed the co-inhibitory receptors PD-1 and TIGIT as well as the activation marker CD38 and showed elevated frequencies of CD39. These results demonstrate that MHC class II tetramer enrichment is a sensitive approach to investigate ex vivo antigen-specific CD4+ T cells in malaria patients that will aid further analysis of the role of CD4+ T cells during malaria.

Acute Malaria Induces PD1+CTLA4+ Effector T Cells with Cell-Extrinsic Suppressor Function.

In acute Plasmodium falciparum (P. falciparum) malaria, the pro- and anti-inflammatory immune pathways must be delicately balanced so that the parasitemia is controlled without inducing immunopathology. An important mechanism to fine-tune T cell responses in the periphery is the induction of coinhibitory receptors such as CTLA4 and PD1. However, their role in acute infections such as P. falciparum malaria remains poorly understood. To test whether coinhibitory receptors modulate CD4+ T cell functions in malaria, blood samples were obtained from patients with acute P. falciparum malaria treated in Germany. Flow cytometric analysis showed a more frequent expression of CTLA4 and PD1 on CD4+ T cells of malaria patients than of healthy control subjects. In vitro stimulation with P. falciparum-infected red blood cells revealed a distinct population of PD1+CTLA4+CD4+ T cells that simultaneously produced IFNγ and IL10. This antigen-specific cytokine production was enhanced by blocking PD1/PDL1 and CTLA4. PD1+CTLA4+CD4+ T cells were further isolated based on surface expression of PD1 and their inhibitory function investigated in-vitro. Isolated PD1+CTLA4+CD4+ T cells suppressed the proliferation of the total CD4+ population in response to anti-CD3/28 and plasmodial antigens in a cell-extrinsic manner. The response to other specific antigens was not suppressed. Thus, acute P. falciparum malaria induces P. falciparum-specific PD1+CTLA4+CD4+ Teffector cells that coproduce IFNγ and IL10, and inhibit other CD4+ T cells. Transient induction of regulatory Teffector cells may be an important mechanism that controls T cell responses and might prevent severe inflammation in patients with malaria and potentially other acute infections.

Human cord blood CD4+CD25hi regulatory T cells suppress prenatally acquired T cell responses to Plasmodium falciparum antigens.

In malaria endemic regions, a fetus is often exposed in utero to Plasmodium falciparum blood-stage Ags. In some newborns, this can result in the induction of immune suppression. We have previously shown these modulated immune responses to persist postnatally, with a subsequent increase in a child's susceptibility to infection. To test the hypothesis that this immune suppression is partially mediated by malaria-specific regulatory T cells (T(regs)) in utero, cord blood mononuclear cells (CBMC) were obtained from 44 Kenyan newborns of women with and without malaria at delivery. CD4(+)CD25(lo) T cells and CD4(+)CD25(hi) FOXP3(+) cells (T(regs)) were enriched from CBMC. T(reg) frequency and HLA-DR expression on T(regs) were significantly greater for Kenyan as compared with North American CBMC (p < 0.01). CBMC/CD4(+) T cells cultured with P. falciparum blood-stage Ags induced production of IFN-γ, IL-13, IL-10, and/or IL-5 in 50% of samples. Partial depletion of CD25(hi) cells augmented the Ag-driven IFN-γ production in 69% of subjects with malaria-specific responses and revealed additional Ag-reactive lymphocytes in previously unresponsive individuals (n = 3). Addition of T(regs) to CD4(+)CD25(lo) cells suppressed spontaneous and malaria Ag-driven production of IFN-γ in a dose-dependent fashion, until production was completely inhibited in most subjects. In contrast, T(regs) only partially suppressed malaria-induced Th2 cytokines. IL-10 or TGF-ß did not mediate this suppression. Thus, prenatal exposure to malaria blood-stage Ags induces T(regs) that primarily suppress Th1-type recall responses to P. falciparum blood-stage Ags. Persistence of these T(regs) postnatally could modify a child's susceptibility to malaria infection and disease.

Atypical memory B-cells are associated with Plasmodium falciparum anemia through anti-phosphatidylserine antibodies.

Anemia is a common complication of malaria that is characterized by the loss of infected and uninfected erythrocytes. In mouse malaria models, clearance of uninfected erythrocytes is promoted by autoimmune anti-phosphatidylserine (PS) antibodies produced by T-bet+B-cells, which bind to exposed PS in erythrocytes, but the mechanism in patients is still unclear. In Plasmodium falciparum patients with anemia, we show that atypical memory FcRL5+T-bet+ B-cells are expanded and associate both with higher levels of anti-PS antibodies in plasma and with the development of anemia in these patients. No association of anti-PS antibodies or anemia with other B-cell subsets and no association of other antibody specificities with FcRL5+T-bet+ B-cells is observed, revealing high specificity in this response. We also identify FcRL5+T-bet+ B-cells as producers of anti-PS antibodies in ex vivo cultures of naïve human peripheral blood mononuclear cells (PBMC) stimulated with P.-falciparum-infected erythrocyte lysates. These data define a crucial role for atypical memory B-cells and anti-PS autoantibodies in human malarial anemia.

Cytotoxic T Cell-Derived Granzyme B Is Increased in Severe Plasmodium Falciparum Malaria.

In Plasmodium falciparum malaria, CD8+ T cells play a double-edged role. Liver-stage specific CD8+ T cells can confer protection, as has been shown in several vaccine studies. Blood-stage specific CD8+ T cells, on the other hand, contribute to the development of cerebral malaria in murine models of malaria. The role of CD8+ T cells in humans during the blood-stage of P. falciparum remains unclear. As part of a cross-sectional malaria study in Ghana, granzyme B levels and CD8+ T cells phenotypes were compared in the peripheral blood of children with complicated malaria, uncomplicated malaria, afebrile but asymptomatically infected children and non-infected children. Granzyme B levels in the plasma were significantly higher in children with febrile malaria than in afebrile children. CD8+ T cells were the main T cell subset expressing granzyme B. The proportion of granzyme B+ CD8+ T cells was significantly higher in children with complicated malaria than in uncomplicated malaria, whereas the activation marker CD38 on CD8+ T cells showed similar expression levels. This suggests a pathogenic role of cytotoxic CD8+ T cells in the development of malaria complications in humans.

Author Correction: Differential expression pattern of co-inhibitory molecules on CD4+ T cells in uncomplicated versus complicated malaria.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Differential expression pattern of co-inhibitory molecules on CD4+ T cells in uncomplicated versus complicated malaria.

The immune response of malaria patients is a main factor influencing the clinical severity of malaria. A tight regulation of the CD4+ T cell response or the induction of tolerance have been proposed to contribute to protection from severe or clinical disease. We therefore compared the CD4+ T cell phenotypes of Ghanaian children with complicated malaria, uncomplicated malaria, asymptomatic Plasmodium falciparum (Pf) infection or no infection. Using flow cytometric analysis and automated multivariate clustering, we characterized the expression of the co-inhibitory molecules CTLA-4, PD-1, Tim-3, and LAG-3 and other molecules implicated in regulatory function on CD4+ T cells. Children with complicated malaria had higher frequencies of CTLA-4+ or PD-1+ CD4+ T cells than children with uncomplicated malaria. Conversely, children with uncomplicated malaria showed a higher proportion of CD4+ T cells expressing CD39 and Granzyme B, compared to children with complicated malaria. In contrast, asymptomatically infected children expressed only low levels of co-inhibitory molecules. Thus, different CD4+ T cell phenotypes are associated with complicated versus uncomplicated malaria, suggesting a two-sided role of CD4+ T cells in malaria pathogenesis and protection. Deciphering the signals that shape the CD4+ T cell phenotype in malaria will be important for new treatment and immunization strategies.

Protein structure shapes immunodominance in the CD4 T cell response to yellow fever vaccination.

The live attenuated yellow fever (YF) vaccine is a highly effective human vaccine and induces long-term protective neutralizing antibodies directed against the viral envelope protein E. The generation of such antibodies requires the help of CD4 T cells which recognize peptides derived from proteins in virus particles internalized and processed by E-specific B cells. The CD4 T helper cell response is restricted to few immunodominant epitopes, but the mechanisms of their selection are largely unknown. Here, we report that CD4 T cell responses elicited by the YF-17D vaccine are focused to hotspots of two helices of the viral capsid protein and to exposed strands and loops of E. We found that the locations of immunodominant epitopes within three-dimensional protein structures exhibit a high degree of overlap between YF virus and the structurally homologous flavivirus tick-borne encephalitis virus, although amino acid sequence identity of the epitope regions is only 15-45%. The restriction of epitopes to exposed E protein surfaces and their strikingly similar positioning within proteins of distantly related flaviviruses are consistent with a strong influence of protein structure that shapes CD4 T cell responses and provide leads for a rational design of immunogens for vaccination.

Rapid-Antigen Test Negative Malaria in a Traveler Returning From Thailand, Molecularly Diagnosed as Plasmodium knowlesi.

Plasmodium knowlesi has been identified in the last decade as a fifth species causing malaria in areas of South East Asia. Due to its short erythrocytic cycle, rapid development of high parasitemia and severe manifestations are frequently observed. Therefore, prompt diagnosis of infection is essential to prevent complications, but the low sensitivity of rapid diagnostic tests for P knowlesi pose a diagnostic challenge in acute settings. In this study, we report the case of a German traveler to Thailand, who was treated for P knowlesi malaria after returning to Germany. Rapid antigen test for malaria was negative on presentation. Diagnosis of a nonfalciparum malaria was made based on microscopy, and species definition was determined using polymerase chain reaction technique.

IL-22 dampens the T cell response in experimental malaria.

A tight regulation between the pro- and anti-inflammatory immune responses during plasmodial infection is of crucial importance, since a disruption leads to severe malaria pathology. IL-22 is a member of the IL-10 cytokine family, which is known to be highly important in immune regulation. We could detect high plasma levels of IL-22 in Plasmodium falciparum malaria as well as in Plasmodium berghei ANKA (PbA)-infected C57BL/6J mice. The deficiency of IL-22 in mice during PbA infection led to an earlier occurrence of cerebral malaria but is associated with a lower parasitemia compared to wt mice. Furthermore, at an early time point of infection T cells from PbA-infected Il22(-/-) mice showed an enhanced IFNγ but a diminished IL-17 production. Moreover, dendritic cells from Il22(-/-) mice expressed a higher amount of the costimulatory ligand CD86 upon infection. This finding can be corroborated in vitro since bone marrow-derived dendritic cells from Il22(-/-) mice are better inducers of an antigen-specific IFNγ response by CD8(+) T cells. Even though there is no IL-22 receptor complex known on hematopoietic cells, our data suggest a link between IL-22 and the adaptive immune system which is currently not identified.

Immunizing school-age children and adolescents: experience from low- and middle-income countries.

OBJECTIVE: Given the increased attention on the need for booster immunizations of older children and adolescents, as well as new primary vaccine series that specifically target school-age children and adolescents, we reviewed the current state of vaccine delivery to school-age children and adolescents in low- and middle-income countries. METHODS: We searched the published literature and unpublished sources for articles, meeting presentations, technical reports and program documents related to immunization policies and programs for school-age children and/or adolescents between 6 and 19 years of age in low- and middle-income countries. FINDINGS: We found several examples of ongoing school-age children and adolescent immunization in low- and middle-income countries. Reasons to vaccinate this age group include vaccines specifically targeted for this age group, waning immunity from prior vaccination, "catch-up" vaccination, acceleration of disease control or elimination efforts, and age distribution shift in the incidence of vaccine-preventable diseases. Multiple delivery strategies are currently in use: routine immunization, supplementary immunization activities, and Child Health Days and similar activities. Vaccines can be delivered in fixed sites, or through outreach. Most immunization programs that target adolescents and school-aged children are providing boosters of infant vaccines at school entry age, with scant experience in delivery of primary vaccination series in adolescents. Few of these programs have been formally evaluated and dissemination of lessons learned is limited. CONCLUSIONS: This baseline description may facilitate immunization program planning in countries considering vaccinating this age group. Additionally, this summary may inform plans for operational research and program evaluation designed to expand vaccine delivery to school-age children and adolescents in low- and middle-income countries.