HTLV-1 targets human placental trophoblasts in seropositive pregnant women

Abstract

Human T cell leukemia virus type 1 (HTLV-1) is mainly transmitted vertically through breast milk. The rate of mother-to-child transmission (MTCT) through formula feeding, although significantly lower than through breastfeeding, is approximately 2.4%-3.6%, suggesting the possibility of alternative transmission routes. MTCT of HTLV-1 might occur through the uterus, birth canal, or placental tissues; the latter is known as transplacental transmission. Here, we found that HTLV-1 proviral DNA was present in the placental villous tissues of the fetuses of nearly half of pregnant carriers and in a small number of cord blood samples. An RNA ISH assay showed that HTLV-1-expressing cells were present in nearly all subjects with HTLV-1-positive placental villous tissues, and their frequency was significantly higher in subjects with HTLV-1-positive cord blood samples. Furthermore, placental villous trophoblasts expressed HTLV-1 receptors and showed increased susceptibility to HTLV-1 infection. In addition, HTLV-1-infected trophoblasts expressed high levels of viral antigens and promoted the de novo infection of target T cells in a humanized mouse model. In summary, during pregnancy of HTLV-1 carriers, HTLV-1 was highly expressed in placental villous tissues, and villous trophoblasts showed high HTLV-1 sensitivity, suggesting that MTCT of HTLV-1 occurs through the placenta.

Keywords: Epidemiology; Infectious disease; Virology.

Figure 1. Comparative analysis of HTLV-1 proviral load in pregnant HTLV-1 carriers.

HTLV screening tests for 254 pregnant HTLV-1 carriers were performed during the second trimester of pregnancy. All donors were serologically positive for HTLV-1 infection. Each dot in the dot plots and scatterplots indicates the HTLV-1 proviral load (PVL) of a single specimen. (A) Detection of HTLV-1 in, and differences in PVL between, maternal blood, placental villous tissue, and cord blood in 254 seropositive women. (B) Comparison of maternal blood PVL among 248 pregnant carriers with PVL in the maternal blood, who were distinguished by proviral DNA detection in the placental villous tissue and/or in the cord blood. In A and B, bold lines indicate the median values of PVL. The P values were calculated with Kruskal-Wallis test followed by Dunn’s multiple-comparisons test. (C–E) Correlation analysis of the PVL was performed. Spearman’s rank correlation test was used to identify statistically significant correlations between values. A positive correlation was detected between PVL in the placental villous tissue and PVL in the maternal blood of 140 pregnant HTLV-1 carriers in whom provirus was detected in the placental villous tissue (C). A nonsignificant correlation was observed between cord blood PVL and maternal blood PVL (D) or placental villous PVL (E) for the 6 pregnant HTLV-1 carriers in whom provirus was detected in the cord blood. (F) Representative electrophoretogram of 6 independent experiments of microsatellite genotyping using short tandem repeat (STR) markers. STR loci of the maternal blood were distinct from those of fetal tissues derived from the same specimen. Amelogenin confirmed the presence of the X chromosome–specific allele alone in the maternal blood, and the X and Y chromosome–specific alleles in the placental villous tissue and the cord blood.

Figure 2. Comparative analysis of anti–HTLV-1 antibody titers in pregnant HTLV-1 carriers.

The anti–HTLV-1 antibody titer was measured in maternal and cord blood samples from 122 subjects using a chemiluminescent enzyme immunoassay (CLEIA). Each dot in the dot plots indicates the HTLV-1 PVL or antibody titer of a single specimen. (A) Relationship between PVL and HTLV-1 antibody titer in the maternal blood measured by CLEIA. (B and C) HTLV-1 antibody titers in maternal blood and cord blood were compared to assess their relationship (B) and their magnitude (C). Bold lines indicate the median values of antibody titers. No significant differences in antibody titer were observed between maternal blood and cord blood (NS by Mann-Whitney U test). Red dots represent pregnant carriers who tested positive for HTLV-1 provirus in the cord blood. In A and B, Spearman’s rank correlation test was used to identify statistically significant correlations between values. COI, cutoff index.

Figure 3. Development of a plus- and minus-strand HTLV-1 mRNA–specific RNAscope ISH assay using a humanized mouse model.

(A) Schematic drawing of the experimental schedule is shown. All NOJ mice were reconstituted with a human immune system by the intrahepatic transplantation of human CD133⁺ hematopoietic stem cells into newborn mice. The mice were inoculated orally with HTLV-1–infected MT-2 cells at 0 days postinfection (dpi) and were sacrificed at 63 dpi (arrows). Arrowheads indicate the time points of blood collection. (B and C) RNA ISH with HTLV-1–specific probes targeting plus-strand mRNAs (B) and minus-strand mRNAs (C) was used to stain the spleen and lymph node sections of mock- or HTLV-1–infected humanized mice. Viral mRNAs were detected using DAB chromogen (brown). Boxes indicate regions shown at a higher magnification in adjacent lower panels. Data are representative of at least 3 nonserial tissue sections. Scale bars in lower and higher magnifications represent 500 μm and 100 μm, respectively.

Figure 4. Detection of HTLV-1 plus-strand mRNAs in pregnant carrier–derived placental villous tissues.

RNA ISH with HTLV-1–specific probes targeting plus-strand mRNAs was used to stain the placental villous tissue sections of pregnant HTLV-1 carriers. The carriers were identical to those listed in Table 2. (A) ISH results are shown for each group of pregnant carriers (nos. 1, 9, and 16), and for a pregnant chronic ATL patient (no. 19). Viral mRNA was detected using DAB chromogen (brown). Boxes indicate regions shown at a higher magnification in adjacent lower panels. Data are representative of at least 5 nonserial tissue sections. Scale bars in lower and higher magnifications represent 200 μm and 50 μm, respectively. (B) Quantitative evaluation of ISH results. The total number of pX probe–positive foci in stained sections was counted by microscopy. Data are expressed as the mean ± SD from 5 nonserial tissue sections. The Mann-Whitney U test was performed to compare statistical differences between groups in subjects with HTLV-1–negative and –positive cord blood samples (nos. 7–12 vs. nos. 13–18).

Figure 5. Evaluation of the susceptibility of human placental cells to HTLV-1 infection.

(A) Surface expression of HTLV-1 receptor molecules on primary placental cells. The expressions of GLUT1, NRP1, and SDC1 were confirmed by immunofluorescence (green and red), and cell nuclei were stained with DAPI (blue). (B) Schematic structure of rVSVs expressing GFP, with or without an HTLV-1 envelope (HTEnv), and the WT construct is shown. (C) HTLV-1 envelope–dependent infection of placental cells. Cells were infected with non–G-complemented VSVΔG/GFP or VSVHTEnv2. At 24 hours postinfection (hpi), GFP expression was evaluated to identify rVSV-infected cells (green) among all cells stained with DAPI (blue). In A and C, stained cells were observed by fluorescence microscopy. Scale bars: 400 μm. (D) Evaluation of viral growth kinetics. In rVSV-infected placental cells, the total number of GFP-positive cells was counted every 24 hours until 72 hpi. The results were calculated as the cell number per square centimeter. (E) Relative expression level of HTLV-1 receptor genes in VTs. Cells were stimulated with IGF-1 or mock-stimulated. The normalized expression levels for mock controls were set as 1 as a reference. (F) Increase in HTLV-1 susceptibility in IGF-1–stimulated VTs. Cells were initially stimulated with IGF-1 or mock-stimulated and then infected with non–G-complemented VSVHTEnv2 in the presence or absence of HTLV-1 envelope–specific neutralizing antibody (LAT-27). The total number of GFP-positive cells was counted, and the relative infectivity was calculated. In D–F, asterisks represent significant differences versus the data for 24 hpi, control, and LAT-27(–), respectively. *P < 0.05, **P < 0.01 by 2-way ANOVA followed by Dunnett’s multiple-comparisons test. Data are means ± SD of 3 independent experiments. VT, villous trophoblasts; VMF, villous mesenchymal fibroblasts; PVEC, placental vascular endothelial cells.

Figure 6. HTLV-1–infected trophoblasts induce de novo infection in humanized mice.

(A) Schematic of the experimental schedule. After humanization, 9 mice were inoculated i.p. with HTLV-1–harboring placental cells at 0 dpi (VT, n = 3; VMF, n = 3; PVEC, n = 3). Arrowheads indicate the time points of blood collection. All mice were observed carefully during the experimental period at 42 dpi. (B) Cell surface expression level of HTLV-1 envelope glycoprotein was analyzed in primary placental cells cocultured with MT-2 cells. The cells were stained with anti–HTLV-1 gp46 mAb or mouse IgG1 mAb as an isotype control. Representative cells (left) and the percentages of HTLV-1 gp46–positive cells (right) are shown. Data are means ± SD of 3 independent experiments. (C) Quantification of HTLV-1 PVL in the peripheral blood of inoculated mice. HTLV-1 PVL was determined by real-time PCR at 14, 28, and 42 dpi. One dot represents the result of an individual mouse. Undetected samples were given an arbitrary value of 10⁰. (D and E) Human CD45⁺ leucocytes, CD3⁺ lymphocytes, total CD4⁺ T cells, and CD25⁺ T cells were routinely analyzed by flow cytometry. One dot represents the result of an individual mouse. The absolute numbers of human CD45⁺ leucocytes are shown in D. Frequencies of lymphocytes positive for the indicated marker are shown in E. CD3⁺ lymphocytes were gated to analyze the populations of CD4⁺ and CD25⁺ T cells. Asterisks represent significant differences versus the data for VTs in B, or PVEC-inoculated mice in C–E. *P < 0.05, **P < 0.01 by 1-way ANOVA followed by Dunnett’s multiple-comparisons test. ND, not detected.

Figure 7. Detection of HTLV-1–infected placental trophoblasts in vivo.

(A) Simultaneous detection of 2 target genes within the same placental villous sections using RNA ISH duplex assay. Probes targeting HTLV-1 minus-strand mRNAs (HBZ) and KRT7, a trophoblast marker, were used to stain the tissue sections of pregnant carriers. The carriers with placental villous PVL (placental villous PVL–positive, and cord blood PVL–negative or –positive) were identical to those in Figure 4 and listed in Table 2. KRT7 was detected using DAB chromogen (brown), and HBZ was detected using fast red chromogen (red). ISH results are shown in the groups of subjects with HTLV-1–negative and –positive cord blood samples (nos. 8 and 10 vs. nos. 13 and 16). (B) RNA ISH combined with immunofluorescence (IF) assay was performed as a confirmatory test. KRT7 protein was detected using FITC (green), HBZ was detected using fast red as a fluorochrome (red), and cell nuclei were stained with NucBlue (blue). Black boxes in A and white boxes in B represent 25 μm squares and 50 μm squares, respectively, and indicate the regions shown at a higher magnification in the same panels. In A and B, data are representative of at least 2 nonserial tissue sections. (C) Quantitative evaluation of RNA ISH combined with IF results. The total number of HBZ probe–positive foci in KRT7-positive or -negative cells of each section was counted by fluorescence microscopy. Data are mean ± SD values from 6 tissue sections of each group. The multiple t test was performed to compare statistical differences between groups in subjects with HTLV-1–negative and –positive cord blood samples (nos. 7–12 vs. nos. 13–18). Asterisks in C represent significant differences between KRT7-negative and KRT7-positive cells; **P < 0.01 by Mann-Whitney U-test.