Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation

Mary Prahl, Prasanna Jagannathan, Tara I McIntyre, Ann Auma, Lila Farrington, Samuel Wamala, Mayimuna Nalubega, Kenneth Musinguzi, Kate Naluwu, Esther Sikyoma, Rachel Budker, Hilary Vance, Pamela Odorizzi, Patience Nayebare, John Ategeka, Abel Kakuru, Diane V Havlir, Moses R Kamya, Grant Dorsey, Margaret E Feeney, Mary Prahl, Prasanna Jagannathan, Tara I McIntyre, Ann Auma, Lila Farrington, Samuel Wamala, Mayimuna Nalubega, Kenneth Musinguzi, Kate Naluwu, Esther Sikyoma, Rachel Budker, Hilary Vance, Pamela Odorizzi, Patience Nayebare, John Ategeka, Abel Kakuru, Diane V Havlir, Moses R Kamya, Grant Dorsey, Margaret E Feeney

Abstract

Background: In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses.

Methods: Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes.

Results: Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12-20 weeks of gestation; p = 0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p = 0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3-CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001).

Conclusion: Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

Keywords: CD4 T cells; Dendritic cells; Fetal immune response; Immune tolerance; Loop-mediated isothermal amplification; Pregnancy-associated malaria.

Figures

Fig. 1
Fig. 1
Flow cytometry gating strategy. Flow cytometric analysis of cord blood live CD4+CD25+ T cells revealed two distinct sub-populations based on FoxP3 and CD127 expression, T regulatory (CD4+CD25+FoxP3−CD127lo/−) and CD127+ (CD4+CD25+FoxP3−CD127hi) cells
Fig. 2
Fig. 2
Infants born to mothers with parasitaemia early in gestation have higher cord blood Treg counts. a Absolute Treg counts were higher in infants born to mothers with parasitaemia at time of enrolment (12–20 weeks gestation) by Wilcoxon ranksum testing (n = 152); b frequency of cord blood Tregs were not significantly associated with malaria exposure outcomes by Wilcoxon rank sum testing (n = 166)
Fig. 3
Fig. 3
Cord blood CD25+FoxP3−CD127+ CD4 T cell frequency is higher in placental Plasmodium LAMP infection. Frequencies of cord blood CD4+CD25+FoxP3−CD127+ cells are higher in infants with positive placental blood LAMP test indicating presence of Plasmodium DNA in placental blood (Student’s t test; n = 166)
Fig. 4
Fig. 4
Memory and activation marker expression by CD127+ CD4 cells. a Expression of CCR7 and CD45RO expression on total CD4 cells, Tregs and CD127+ CD4 cells; b percentage of CD4+ T cells, Tregs, and CD127+ CD4 cells that are phenotypically non-naïve by CCR7/CD45RO gating (n = 166); c percentage of Tregs vs CD127+ cells expressing cell surface markers (n = 166); d distinct expression of cell surface markers CCR4, CCR7, and CD95 by all CD4+CD25+ T cells, Tregs, and CD127+ cells in one representative cord blood sample
Fig. 5
Fig. 5
Myeloid dendritic cell counts are higher in placental histopathologic malaria infection. Absolute counts of total dendritic cells (Lin−HLA-DR+) (DCs), myeloid dendritic cells (Lin−HLA-DR+CD11c+CD123−) (mDCs), and plasmacytoid dendritic cells (Lin-HLA-DR+CD11c−CD123+) (pDCs) in cord blood of infants with negative and positive histopathology for placental malaria (Wilcoxon rank sum; n = 145)

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