Plasmodium falciparum exposure in utero, maternal age and parity influence the innate activation of foetal antigen presenting cells

Background: Malaria in pregnancy is associated with immunological abnormalities in the newborns, such as hampered T-helper 1 responses and increased T-regulatory responses, while the effect of maternal Plasmodium falciparum infection on foetal innate immunity is still controversial. Materials and methods: The immunophenotype and cytokine release by dendritic cells (DC) and monocytes were evaluated in cord blood from 59 Beninese women with or without malaria infection by using flow cytometry. Results: Accumulation of malaria pigment in placenta was associated with a partial maturation of cord blood myeloid and plasmacytoid DC, as reflected by an up-regulated expression of the major histocompatibility complex class II molecules, but not CD86 molecules. Cells of newborns of mothers with malaria pigment in their placenta also exhibited significantly increased cytokine responses upon TLR9 stimulation. In addition, maternal age and parity influenced the absolute numbers and activation status of cord blood antigen-presenting cells. Lastly, maternal age, but not parity, influenced TLR3, 4 and 9 responses in cord blood cells. Discussion: Our findings support the view that placental parasitization, as indicated by the presence of malaria pigment in placental leukocytes, is significantly associated with partial maturation of different DC subsets and also to slightly increased responses to TLR9 ligand in cord blood. Additionally, other factors, such as maternal age and parity should be taken into consideration when analysing foetal/neonatal innate immune responses. Conclusion: These data advocate a possible mechanism by which PAM may modulate foetal/neonatal innate immunity

Protection by and maintenance of CD4 effector memory and effector T cell subsets in persistent malaria infection

<div><p>Protection at the peak of <i>Plasmodium chabaudi</i> blood-stage malaria infection is provided by CD4 T cells. We have shown that an increase in Th1 cells also correlates with protection during the persistent phase of malaria; however, it is unclear how these T cells are maintained. Persistent malaria infection promotes protection and generates both effector T cells (Teff), and effector memory T cells (Tem). We have previously defined new CD4 Teff (IL-7Rα^-) subsets from Early (Teff^Early, CD62L^hiCD27⁺) to Late (Teff^Late, CD62L^loCD27^-) activation states. Here, we tested these effector and memory T cell subsets for their ability to survive and protect <i>in vivo</i>. We found that both polyclonal and <i>P</i>. <i>chabaudi</i> Merozoite Surface Protein-1 (MSP-1)-specific B5 TCR transgenic Tem survive better than Teff. Surprisingly, as Tem are associated with antigen persistence, Tem survive well even after clearance of infection. As previously shown during T cell contraction, Teff^Early, which can generate Tem, also survive better than other Teff subsets in uninfected recipients. Two other Tem survival mechanisms identified here are that low-level chronic infection promotes Tem both by driving their proliferation, and by programming production of Tem from Tcm. Protective CD4 T cell phenotypes have not been precisely determined in malaria, or other persistent infections. Therefore, we tested purified memory (Tmem) and Teff subsets in protection from peak pathology and parasitemia in immunocompromised recipient mice. Strikingly, among Tmem (IL-7Rα^hi) subsets, only Tem^Late (CD62L^loCD27^-) reduced peak parasitemia (19%), though the dominant memory subset is Tem^Early, which is not protective. In contrast, all Teff subsets reduced peak parasitemia by more than half, and mature Teff can generate Tem, though less. In summary, we have elucidated four mechanisms of Tem maintenance, and identified two long-lived T cell subsets (Tem^Late, Teff^Early) that may represent correlates of protection or a target for longer-lived vaccine-induced protection against malaria blood-stages.</p></div

Persistent infection promotes Tem survival.

<p><b>A)</b> Schematic representation of the experimental design. Memory T cell subsets were sorted from infected B5 TCR Tg mice (d60 p.i.) and the same number of each subset (2.5 x 10⁵) were transferred into either infection-matched (d60 p.i., top) or uninfected (bottom) Thy1.1 hosts for 60 days after transfer. <b>B)</b> Graph shows numbers of recovered B5 T cells (CD4⁺ Thy1.2⁺) in recipients of Tem^Early. <b>C)</b> Concatenated contour plots show division (CFSE-), and levels of CD127, and phenotypes (CD62L, CD27) of cells recovered from Tem^Early recipients. Summary bar graphs show average of divided cells or fraction in each Tmem subset gate on recovery. <b>D)</b> Graph shows numbers of recovered B5 T cells in recipients of Tem^Late. <b>E)</b> Concatenated contour plots show levels of CFSE, and CD127, and phenotypes (CD62L, CD27) of cells recovered from Tem^Late recipients. Summary bar graphs show average of divided cells or fraction in each Tmem subset gate on recovery. Data are representative of 2–4 mice per group from 2 similar experiments. Data was analyzed by Student’s <i>t</i> test, and error bar represents SEM, *p<0.05, n.s.–not significant. On summary bar graphs symbols refer to differences between fractions of recovered cells in each Tmem subset in a given stacked bar, one symbol = p<0.05; two symbols, **p<0.01 comparing Tcm to TemL; three symbols, ***p<0.001 comparing Tem^Early to Tem^Late, ^{<b><i>#</i></b>} comparing Tcm (very few) to Tem^Early, ^<b>+</b> comparing Tem^Early to Tem^Late.</p

All Effector T cell subsets protect from parasitemia.

<p><b>A)</b> Schematic of experimental model. Effector T cell subsets were sorted from spleens of B5 TCR Tg on d8 p.i., and transferred (5x10⁵) with immune CD19⁺ BALB/c B cells (1x10⁷) into RAG2^o mice that were then infected with <i>P</i>. <i>chabaudi</i> (5x10⁴ iRBC). Parasitemia and pathology were followed for two weeks. Graphs showing average <b>B)</b> peak parasitemia (%iRBC/RBC) summarized from two experiments (n = 6), <b>C)</b> % change of weight, and hypothermia of recipient mice at the peak of each symptom for each recipient (d8-10 p.i.). Flow cytometry of splenocytes was done on day 14 p.i. and <b>D)</b> graph shows average number of B5 T cells (CD4⁺Thy1.2⁺) recovered. <b>E)</b> Histograms, contour overlay (Teff^Late), and summary graphs of cytokines produced by T cells from recipients of each Teff subset. Data show 2–3 mice per group and are representative of 3 independent experiments. Error bars show SEM, *p<0.05, **p<0.01, ***p<0.001, n.s–not significant.</p

Mature effector T cells decay faster than effector memory T cells in <i>P</i>. <i>chabaudi</i>.

<p>Decay of malaria-specific polyclonal cells was detected by infecting C57Bl/6 mice with <i>P</i>. <i>chabaudi</i> and administration of BrdU either (<b>A-E</b>) during the memory phase (days 24–30), or (<b>F-K</b>) the peak of infection (days 4–10) to label Teff. The day after the end of BrdU administration, the infection was terminated with mefloquine (MQ), and decay of CD4⁺ memory (CD44^hi CD127^hi CD11a⁺, BrdU^+/-) and effector (CD127^<b>-</b>, (CD11a, BrdU)^+/-) T cells in the spleen was determined by flow cytometry. <b>A)</b> Schematic representation of the experimental design for memory phase. <b>B)</b> Plots show the gating strategy for Tmem, including BrdU. Graphs showing <b>C)</b> number of Tmem (CD4⁺ CD11a⁺ CD44^hi CD127^hi) and <b>D)</b> survival of memory T cell subsets in the spleen after parasite clearance. <b>E)</b> Graphs showing percentage (left) and number (right) of cells in each Tmem subset that survive after proliferating days 24–30 (BrdU⁺). <b>F)</b> Schematic representation of the experimental design to study decay of effector T cells, where BrdU was given days 4–10 p.i., and infection was terminated with MQ days 10–14 p.i. Flow cytometric gating and graphs showing <b>G)</b> number of Teff (CD4⁺ CD127^<b>-</b>) and <b>H)</b> survival of Teff subset populations in the spleen after parasite clearance. <b>I)</b> Plots showing the gating strategy for Teff from CD11a+ to Teff (CD4⁺ CD11a⁺ CD44^hi CD127^lo). Graphs showing <b>J)</b> the number of divided Teff (CD4⁺ CD11a⁺ CD127^- BrdU⁺), as they decay after labelling days 4–10 p.i, and subsequent parasite clearance, and <b>K)</b> the percentage (left) and number (right) of cells in each of the divided Teff subsets as they decay. Data represent 3 mice per group. Data was analyzed by Student’s <i>t</i> test and error bars represent SEM. * represents a significant difference between subsets at one timepoint. † represents a significant difference between timepoints d10-d30, or d30-50, # from d20-30 only; one symbol p<0.05, two symbols p<0.01, n.s.–not significant with color coding of symbols to indicate which subset changes.</p

Teff^Early survive like Tmem cells, while highly activated Teff subsets decay.

<p><b>A)</b> Schematic representation of the experimental design. T cell subsets were sorted from spleens of infected B5 TCR Tg animals (Effector on d8 p.i. (top) and Memory on d60 p.i. (bottom)) and the same number of T cells of each subset (5 x 10⁴) were transferred into uninfected congenic recipients (Thy1.1) for 60 days. <b>B)</b> Graph showing numbers of B5 TCR Tg (CD4⁺ Thy1.2⁺) T cells recovered from spleens of recipients of each T cell subset on d60 post-transfer. Data represent 4–9 mice per group from two experiments. Data were analyzed using Student’s <i>t</i> test, **p<0.01, *p<0.05. n.s.–not significant. <b>C)</b> Concatenated contour plot of B5 T cells recovered from Teff^Early recipients day 60 post-transfer showing memory (CD44, CD127) and memory T cell subset (CD62L, CD27) phenotype, and summary graph of Tmem phenotype of recovered T cells from the groups of mice that received each Teff subset. Error bars represent SEM.</p

Proposed model of T cell activation and memory T cell differentiation.

<p>Upon activation of naïve T cells, Teff^Early (CD127^- CD62L^hi CD27⁺) are generated, which contain precursors to both effector and memory T cells. Depending on the presence of antigen (or leukopenia) in their environment, they can progress toward maturation of effector T cells, Teff^Int, Teff^Late subsets, or in an uninfected environment, towards differentiation into Tcm and then Tem subsets, the latter being promoted by low-level chronicity. Upon downregulation of CD62L, Teff lose survival potential and become terminal Teff; however, surviving mature Teff can become CD127^hi, and expand when re-activated and promote protection. Tmem can proliferate without downregulating CD127 in conditions of low antigen exposure (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006960#ppat.1006960.g004" target="_blank">Fig 4B</a></b>). Dotted arrows show less common events, or those dependent on environmental changes. Tmem can become Teff again in conditions of re-exposure to higher antigen loads (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006960#ppat.1006960.s002" target="_blank">S2B Fig</a></b>). We have also observed the more terminally differentiated subsets re-expressing CD62L, suggesting plasticity in this process. The degree of differentiation to central or effector memory T cells from Teff^Early is determined in the first week of <i>P</i>. <i>chabaudi</i> infection [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006960#ppat.1006960.ref018" target="_blank">18</a>]. In chronic infection, and for a period after exposure to long-lasting infection, Tcm can continue to generate Tem. Populations that protect best are marked by green asterisks, and populations that survive best are marked by red asterisks.</p

Rested effector T cells protect from parasitemia and pathology.

<p><b>A)</b> Schematic of the experimental design. Effector T cell subsets were sorted from the spleens of B5 TCR Tg at d8 p.i. and transferred (5 x 10⁵) into RAG2^o mice for 60 days before infection. Recipients were then infected with 10⁵ <i>P</i>. <i>chabaudi</i>. Parasitemia, weight, and temperature change was measured for 14 days after infection. <b>B)</b> Average peak parasitemia (%iRBC/RBC) of each mouse (d8-10 p.i.) is shown. As measurements of pathology, the average of <b>C)</b> weight and <b>D)</b> temperature loss (% change) of each recipient at the peak of pathology (d8-10 p.i.) are shown. Flow cytometry was performed to quantitate <b>E)</b> the number of B5 T cells (CD4⁺Thy1.2⁺) recovered from RAG2^o recipients on day 14 p.i. and <b>F)</b> graph showing MFI of IFN-γ in T cells recovered from each group. Data represent 2–3 mice per group and are representative of 3 similar independent experiments. Parasitemia was analyzed using one-way ANOVA and Tukey’s post-hoc. Error bar represents SEM, * p<0.05, ** p<0.01, *** p<0.001, n.s—not significant.</p