Research ArticleMalaria

In utero priming of highly functional effector T cell responses to human malaria

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Science Translational Medicine  17 Oct 2018:
Vol. 10, Issue 463, eaat6176
DOI: 10.1126/scitranslmed.aat6176
  • Fig. 1 Fetal effector memory CD4+ T cell differentiation in cord blood of malaria-exposed infants.

    (A) Representative flow plots of CD4+ T cell effector memory subsets. TCM, CD45RACCR7+; TEM, CD45RACCR7; and TEMRA, CD45RA+CCR7. Values indicate % of CD4+ T cells. (B) Quantification of CD4+ T cell subsets in infants born to mothers with (gray bar; n = 14) or without (white bar; n = 55) active PM. (C) Frequency of TEM cells in infants born to mothers without malaria during pregnancy (white bar; n = 10), with past malaria infection (striped bar; n = 45), or with active PM (gray bar; n = 14). ns, not significant. (D) Representative histograms of CD4+ T cell subset phenotype from a representative PM-exposed infant. TCTL, cytotoxic T lymphocyte; cTFH, circulating T follicular helper cell. (E) Quantification of % positive for phenotypic markers on CD4+ TEM cells in active PM–exposed (gray bar; n = 14) versus unexposed (white bar; n = 55) infants. (F) Gating strategy for CD4+ TH subsets; representative of an active PM–exposed infant. (G) Quantification of CD4+ TH subsets (% of non-naïve CD4+ T cells) in infants born to mothers with (gray bar; n = 20) versus without (white bar; n = 154) active PM. (H) Representative flow plots of Ki67 and CD45RA expression. Values indicate % Ki67+ cells of non-naïve CD4+ T cells (CD45RA). (I) Quantification of non-naïve Ki67+ T cells in active PM–exposed (gray bar; n = 11) versus unexposed (white bar; n = 57) infants. All representative flow plots were gated on live CD14CD19γδTCRCD3+CD8CD4+ T cells. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis and Wilcoxon rank sum test.

  • Fig. 2 Inflammatory cytokine production by cord blood CD4+ and CD8+ T cells from PM-exposed infants.

    (A) Representative flow plots of ICS for production of IFN-γ and TNF-α by non-naïve CD4+ T cells after 5-hour in vitro stimulation of CBMCs with medium alone (upper) or PMA/ionomycin (lower). Gated on live CD14CD19γδTCRCD3+CD8CD4+CD45RA T cells. Values indicate the frequency of cytokine-positive cells within the non-naïve population. (B) Quantification of cytokine-producing cells in infants born to mothers without malaria during pregnancy (white bar; n = 16), with past malaria infection (striped bar; n = 83), or with active PM (gray bar; n = 21). (C) Representative flow plots of ICS for production of IFN-γ and TNF-α by non-naïve CD8+ T cells after 5-hour in vitro stimulation of CBMCs with medium alone (upper) or PMA/ionomycin (lower). Gated on live CD14CD19γδTCRCD3+CD4CD8+CD45RA T cells. Values indicate the frequency of cytokine-positive cells within the non-naïve population. (D) Quantification of cytokine-producing cells in infants born to mothers without malaria during pregnancy (white bar; n = 16), with past malaria infection (striped bar; n = 83), or with active PM (gray bar; n = 21). Values calculated by background subtraction of medium-alone controls. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis and Wilcoxon rank sum test.

  • Fig. 3 Proliferation of cord blood CD4+ and CD8+ T cells in response to malaria antigens.

    (A) Representative CFSE dilution profiles for CD4+ (upper) and CD8+ (lower) T cells after 6-day PfSE (red histogram) or uRBC (gray histogram) stimulation. Values indicate frequency of CFSElow T cells (% of CD4+ or CD8+ T cells). (B) Quantification of CFSElow CD4+ (left) or CD8+ (right) T cells in infants born to mothers without malaria during pregnancy (white bar; n = 11), with past malaria infection (striped bar; n = 69), or with active PM (gray bar; n = 18). (C) CFSElow CD4+ (left) and CD8+ (right) T cell frequencies after 6 days of PfSE stimulation in the presence or absence of αMHC-II or αMHC-I (n = 8). Data are representative of three independent experiments. (D) ICS for cytokine production by CFSE-labeled Pf SE-stimulated CBMCs restimulated on day 6 with PMA/ionomycin in infants born to mothers with (gray bar) or without (white bar) active PM. (E) Quantification of CFSElow cytokine-producing CD4+ and CD8+ T cells stratified by infant malaria exposure (active PM, white bars, n = 79; active PM+, gray bars, n = 17). (F) Transcription factor mRNA expression in sorted CFSElow CD4+ (left) and CD8+ T cells (right) from infants born to active PM+ mothers (n = 3 to 5). Data are representative of three independent experiments. (G) CFSElow CD4+ T cell frequencies in the presence or absence of Tregs. Data are representative of three independent experiments. All representative flow plots were gated on live CD14CD19γδTCRCD3+ T cells. CFSElow frequencies were calculated by background subtraction of uRBC-stimulated and medium-alone controls. *P < 0.05, **P < 0.01, ***P < 0.001, significance assessed by Kruskal-Wallis and Wilcoxon rank sum test for all experiments except MHC blockade and Treg depletion, which were assessed by Wilcoxon matched-pairs signed-rank test.

  • Fig. 4 Proliferation of cord blood CD4+ and CD8+ T cells in response to MSP1 peptide stimulation.

    (A) Representative CFSE dilution profiles for CD4+ (upper) and CD8+ (lower) T cells stimulated with MSP1 peptide pools for 6 days and restimulated with PMA/ionomycin. Gated on live CD14CD19γδTCRCD3+ T cells. Values indicate frequency of CFSElow T cells as % CD4+ or CD8+ T cells. (B) Frequency of CFSElow CD4+ (left) or CD8+ (right) T cells in infants born to mothers without malaria during pregnancy (white bar; n = 4), with past malaria infection (striped bar; n = 38), or with active PM (gray bar; n = 9). (C) Quantification of CFSElow cytokine-producing CD4+ and CD8+ T cells stratified by infant malaria exposure (PM, white bars, n = 41; PM+, gray bars, n = 8). For all experiments, values are calculated by background subtraction of dimethyl sulfoxide–stimulated and medium-alone controls. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis and Wilcoxon rank sum test.

  • Table 1 CD4+ T cell proliferation in response to malaria antigens associated with protection from childhood malaria.

    PY, person years of observation.

    Incidence of clinical malaria through 24 months of age
    % Pf SE-specific
    CFSElow CD4+ T cells
    nEpisodesPYIncidenceIRR (95% CI)P value
    Low (<15%)111621.00.76Reference
    Intermediate (15 to 30%)353662.50.580.79 (0.30–2.06)0.63
    High (≥30%)271048.30.210.28 (0.09–0.84)0.02
    Time to first episode of malaria after birth
    nCumulative risk (95% CI)HR (95% CI)P value
    Low (<15%)1179.6% (51.8–96.8%)Reference
    Intermediate (15 to 30%)3541.1% (26.3–60.1%)0.40 (0.16–0.98)0.05
    High (≥30%)2733.8% (18.5–56.5%)0.28 (0.11–0.77)0.01
    Detection of malaria parasites at routine monthly visits between 2 and 24 months of age*
    n/N%PRR (95% CI)P value
    Low (<15%)25/2699.29%Reference
    Intermediate (15 to 30%)56/7977.03%0.80 (0.33–1.93)0.63
    High (≥30%)19/6203.06%0.33 (0.13–0.89)0.03

    *By blood smear and/or LAMP.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/463/eaat6176/DC1

      Materials and Methods

      Fig. S1. Outline of inclusion criteria and experimental prioritization of cord blood samples.

      Fig. S2. Fetal effector memory CD4+ T cell frequencies are altered by parasites in the placenta but not by maternal gravidity or IPT arm.

      Fig. S3. Fetal effector and memory CD8+ T cell subsets do not differ with PM exposure.

      Fig. S4. Cord blood CD4+ T cell responses are fetal in origin.

      Fig. S5. Cord blood CD4+ T cell responses are transcriptionally heterogeneous.

      Fig. S6. Cytokine production by cord blood CD4+ and CD8+ T cells.

      Fig. S7. Characterization of cord blood CD4+ and CD8+ T cell proliferation in response to malaria antigens.

      Fig. S8. Gating strategy for CD4+ and CD8+ T cell populations.

      Table S1. CD4+ T cell proliferation in response to malaria antigens is associated with protection from childhood malaria with adjustment for maternal malaria exposure.

      Table S2. Details of clinical cohort.

      Table S3. Details of flow cytometric antibodies.

      Table S4. Primary data.

    • The PDF file includes:

      • Materials and Methods
      • Fig. S1. Outline of inclusion criteria and experimental prioritization of cord blood samples.
      • Fig. S2. Fetal effector memory CD4+ T cell frequencies are altered by parasites in the placenta but not by maternal gravidity or IPT arm.
      • Fig. S3. Fetal effector and memory CD8+ T cell subsets do not differ with PM exposure.
      • Fig. S4. Cord blood CD4+ T cell responses are fetal in origin.
      • Fig. S5. Cord blood CD4+ T cell responses are transcriptionally heterogeneous.
      • Fig. S6. Cytokine production by cord blood CD4+ and CD8+ T cells.
      • Fig. S7. Characterization of cord blood CD4+ and CD8+ T cell proliferation in response to malaria antigens.
      • Fig. S8. Gating strategy for CD4+ and CD8+ T cell populations.
      • Table S1. CD4+ T cell proliferation in response to malaria antigens is associated with protection from childhood malaria with adjustment for maternal malaria exposure.
      • Table S2. Details of clinical cohort.
      • Table S3. Details of flow cytometric antibodies.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S4 (Microsoft Excel format). Primary data.

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