Pregnancy-induced transfer of pathogen-specific T cells from mother to fetus in mice

Abstract

Neonatal health is determined by the transfer of maternal antibodies from the mother to the fetus. Besides antibodies, maternal cells cross the placental barrier and seed into fetal organs. Contrary to maternal antibodies, maternal microchimeric cells (MMc) show a high longevity, as they can persist in the offspring until adulthood. Recent evidence highlights that MMc leukocytes promote neonatal immunity against early-life infections in mice and humans. As shown in mice, this promotion of immunity was attributable to an improved fetal immune development. Besides this indirect effect, MMc may be pathogen-specific and thus, directly clear pathogen threats in the offspring postnatally. By using ovalbumin recombinant Listeria monocytogenes (LmOVA), we here provide evidence that OVA-specific T cells are transferred from the mother to the fetus, which is associated with increased activation of T cells and a milder course of postnatal infection in the offspring. Our data highlight that maternally-derived passive immunity of the neonate is not limited to antibodies, as MMc have the potential to transfer immune memory between generations.

Keywords: immunological memory; intrauterine transfer; maternal microchimerism; pregnancy.

Figure 1. LmOVA infection prior to pregnancy induces the accumulation of CD4⁺ and CD8⁺ T cells in the uterus

1. A

   Experimental approach.

2. B

   Numbers of CD4⁺ and CD8⁺ in 1 × 10⁶ uterine cells on gd 18.5 after preconceptual infection of the mother in comparison with non‐infected mothers (n = 7, n = 9, n: biological replicates). T cells were identified as CD45⁺CD3⁺ and either CD4⁺ or CD8⁺ cells.

3. C

   Representative dot plots of CD8⁺ T cells from peripheral blood divided in CD44⁺CD62L⁻ effector/effector memory (EM), CD44⁺CD62L⁺ central memory (CM), and CD44⁻CD62L⁺ naïve subpopulations. Left: non‐infected pregnant control mouse, right: previously infected pregnant mouse.

4. D–G

   Percentage of EM T cells (CD44⁺, CD62L⁻) among CD8⁺ T cells; (D) peripheral blood (n = 6, n = 9), (E) uterus‐draining lymph nodes (n = 6, n = 9), (F) spleen (n = 7, n = 8), (G) uterus (n = 7 each); n: biological replicates.

5. H

   Representative dot plots of ovalbumin‐specific CD8⁺ EM T cells stained with H‐2K^b ovalbumin_257‐264 tetramers (OVA tetramers) from peripheral blood. Left: non‐infected pregnant control mouse, right: previously infected pregnant mouse.

6. I–L

   Percentage of ovalbumin‐specific CD8⁺ EM T cells; (I) peripheral blood (n = 7, n = 9), (J) uterus‐draining lymph nodes (n = 7, n = 9), (K) spleen (n = 8 each), (L) uterus (n = 6, n = 9); n: biological replicates.

Data information: In (B), (D–G), (I–L), data are presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001 (E, J, K, L: Student's t‐test; B, D, F, G, I: Mann–Whitney‐U test). Source data are available online for this figure.

Figure EV1. Composition of CD8⁺ T cell subpopulations after preconceptual infection

1. A

   Gating strategy of CD8⁺ T effector/effector memory cells. Blood of pregnant mice at gd 18.5 after preconceptual infection.

2. B

   Number of CD45⁺ cells in 1 × 10⁶ uterine cells on gd 18.5 after preconceptual infection of mothers in comparison with naïve mothers (n = 7 each, n: biological replicates).

3. C–F

   Percentage of CD8⁺ T cell subpopulations: CD44⁺CD62L⁻ effector/effector memory (EM), CD44⁺CD62L⁺ central memory (CM), CD44⁻CD62L⁺ naïve; (C) peripheral blood (n = 6, n = 9), (D) uterus‐draining lymph nodes (n = 6, n = 9), (E) spleen (n = 8, n = 7), (F) uterus (n = 7 each); n: biological replicates.

Data information: In (B), data are presented as mean ± SEM. In (C–F), data are presented as mean (B, D: Student's t‐test; C, E, F: Mann–Whitney‐U test).

Figure 2. Intergenerational transfer of OVA‐specific MMc from mother to offspring

1. A

   Infection and mating strategy and identification strategy for maternal microchimeric cells (MMc) in offspring of CD45.2⁺ H‐2D^b+ C57BL/6 females mated with CD45.1⁺ H‐2D^d+ Balb/c males.

2. B

   Representative dot plots of column bound ‘non‐MMc’ fraction (upper) and ‘MMc‐enriched’ flow‐through fraction (lower) after negative selection of MMc via antibody labeling of paternal H‐2D^d and magnetic‐activated cell sorting (MACS) gated on CD45.2⁺CD45.1⁻ cells.

3. C–E

   Percentage of CD3⁺ T cells of total MMc; (C) fetal bone marrow (n = 13, n = 14), (D) fetal spleen (n = 12, n = 13), (E) fetal liver (n = 13, n = 14); n: biological replicates.

4. F

   Representative dot plots for OVA‐tetramer staining of CD3⁺ MMc from spleens of fetuses of previously infected or non‐infected mothers.

5. G–I

   Percentage of OVA‐specific MMc stained with OVA tetramers among CD3⁺ microchimeric T cells; (G) bone marrow (n = 13 each), (H) spleen (n = 12 each), (I) liver (n = 11, n = 12); n: biological replicates.

6. J

   Serum anti‐Lm IgG titer of mothers and fetuses at gd 18.5 after preconceptual infection of the mother with LmOVA; n = 3 biological replicates, each averaged from n = 2 technical replicates).

Data information: In (C–E), (G–J), data are presented as mean ± SEM. *P ≤ 0.05 (C, D, E, H: Student's t‐test; G, I: Mann–Whitney‐U). Source data are available online for this figure.

Figure EV2. No differences in pregnancy outcome and MMc numbers in fetal organs after preconceptual infection of female mice

1. A–D

   Pregnancy outcome parameter: (B) Percentage of fetal loss rate (n = 8, n = 9); (C) Number of implantations on gd 18.5 (n = 7, n = 9); (D) Male/female ratio of offspring (n = 6, n = 7); (E) Fetal weight on gd 18.5 (n = 54, n = 55); n: biological replicates.

2. E–G

   Placental evaluation: (E) Area of placental labyrinth (L) (n = 12 each); (F) Area of placental junctional zone (JZ) (n = 12–13); (G) Ratio of placental labyrinth to the junctional zone (L/JZ ratio) as an indicator for placental function on gd 18.5 (n = 12 each); n: biological replicates.

3. H

   Representative depiction of murine placenta assessment, highlighted for labyrinth (L, white) and labyrinth + junctional zone (black). Scale bar = 1,000 μm.

4. I

   Gating strategy of ‘MMc‐enriched’ flow‐through. Bone marrow of fetal mice at gd 18.5 born to a preconceptually infected mother.

5. J–L

   Numbers of MMc in 1 × 10⁶ fetal cells on gd 18.5; (J) bone marrow (n = 14, n = 13), (K) spleen (n = 14, n = 12), (L) liver (n = 13, n = 12); n: biological replicates.

Data information: In (B–H), (J–L), data are presented as mean ± SEM. ****P ≤ 0.0001 (C, E, G, K, L: Student's t‐test; A, B, D, F, J: Mann–Whitney‐U test).

Figure 3. No fetal innate immune activation after preconceptual infection and re‐infection of pregnant mice

1. A

   Experimental approach.

2. B–F

   Percentages of innate immune cell subsets in the fetal spleen; (B) CD45⁺ leukocytes (n = 20, n = 11), (C) CD45⁺CD11b⁺ myeloid cells (n = 19, n = 11), (D) CD45⁺CD11b⁺Gr‐1⁻ monocytes (n = 19, n = 11), (E) CD45⁺CD11b⁺Gr‐1⁺ neutrophils (n = 19, n = 11) and (F) CD45⁺CD11b⁺F4/80⁺ macrophages (n = 20, n = 11); n: biological replicates.

3. G–K

   Percentages of innate immune cell subsets in the fetal liver; (G) CD45⁺ leukocytes (n = 20, n = 10), (H) CD45⁺CD11b⁺ myeloid cells (n = 20, n = 10), (I) CD45⁺CD11b⁺Gr‐1⁻ monocytes (n = 20, n = 10), (J) CD45⁺CD11b⁺Gr‐1⁺ neutrophils (n = 20, n = 10) and (K) CD45⁺CD11b⁺F4/80⁺ macrophages (n = 20, n = 10); n: biological replicates.

Data information: In (B–K), data are presented as mean ± SEM. *P ≤ 0.05 (B, C, G–I: Ordinary one‐way ANOVA; D‐F, K, J: Kruskal–Wallis test). Source data are available online for this figure.

Figure 4. Offspring born to a preconceptually infected mother are less susceptible to infection and show activation of CD3⁺ T cells

1. A

   Experimental approach.

2. B, C

   Neonatal body weight in mg (n = 20–55). (B) Day 7 after birth (day of infection), (C) day 14 after birth (7 days after infection); n: biological replicates.

3. D, E

   Percentages of OVA tetramer⁺ cells among CD3⁺ MMc (percentages of OVA tetramer⁺ among CD3⁺ MMc at gd 18.5 (Fig 2H and I) are included for direct comparison); (D) spleen (n = 5–10), (E) liver (n = 5–10); n: biological replicates.

4. F–I

   Percentage of (F) CD3⁺ T cells of CD45⁺ spleen cells (n = 6–14), (G) OVA‐specific cells of CD3⁺ T cells from spleen cells (n = 6–14), (H) CD11b⁺ cells of CD3⁺ T cells from spleen cells (n = 6–14), and (I) CD11c⁺ cells of CD3⁺ T cells from spleen cells (n = 6–14); n: biological replicates.

Data information: In (B–I), data are presented as mean ± SEM. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001 (B–D, G: Ordinary one‐way ANOVA; E, F, H, I: Kruskal–Wallis test). Source data are available online for this figure.

Figure EV3. Infection alters neonatal weight but does not influence MMc numbers

1. A

   Body weight development after infection of neonates depicted as weight in mg.

2. B, C

   Neonatal body weight in mg (n = 20–55, n: biological replicates). (B) Day 9 after birth (2 days after infection), (C) day 12 after birth (5 days after infection).

3. D, E

   Number of MMc in 1 × 10⁶ fetal cells on day 7 after infection; (D) spleen (n = 6–14), (E) liver (n = 6–11); n: biological replicates.

4. F, G

   Percentage of CD3⁺ T cells among MMc; (F) spleen (n = 6–13), (G) liver (n = 6–12); n: biological replicates.

Data information: In (B–G), data are presented as mean ± SEM. **P ≤ 0.01; ****P ≤ 0.0001 (B, F, G: Ordinary one‐way ANOVA; C–E: Kruskal–Wallis test). Source data are available online for this figure.