Long-Term Programming of CD8 T Cell Immunity by Perinatal Exposure to Glucocorticoids

Abstract

Early life environmental exposure, particularly during perinatal period, can have a life-long impact on organismal development and physiology. The biological rationale for this phenomenon is to promote physiological adaptations to the anticipated environment based on early life experience. However, perinatal exposure to adverse environments can also be associated with adult-onset disorders. Multiple environmental stressors induce glucocorticoids, which prompted us to investigate their role in developmental programming. Here, we report that perinatal glucocorticoid exposure had long-term consequences and resulted in diminished CD8 T cell response in adulthood and impaired control of tumor growth and bacterial infection. We found that perinatal glucocorticoid exposure resulted in persistent alteration of the hypothalamic-pituitary-adrenal (HPA) axis. Consequently, the level of the hormone in adults was significantly reduced, resulting in decreased CD8 T cell function. Our study thus demonstrates that perinatal stress can have long-term consequences on CD8 T cell immunity by altering HPA axis activity.

Keywords: CD8 T cells; developmental plasticity; early-life stress; glucocorticoids; perinatal programming.

Figure 1.. Perinatal GC exposure persistently decreased CD8 T cell immune function in adulthood.

(A) Illustration of perinatal DEX treatment model. (B-F) Footpads of 12 week old B6 mice were immunized with PBS+OVA or LPS+IFA+OVA. Draining LNs were collected post immunization, and cells were re-stimulated with OVA. Illustration of footpad immunization experimental model (B). IFN-γ+ protein production (72 h OVA stimulation) was analyzed by ELISA (Ctrl: Control, n=8–9/group (female and male)) (C). Intracellular IFN-γ+ protein analysis by flow cytometry (24h OVA stimulation, n=8–10/group, (female and male)) (D). Flow cytometric analysis of IFN-γ+ CD4+ cells and IFN-γ+ CD8+ cells (n=5–7/group, male and female) (E). Flow cytometric analysis of CD44+ IFN-γ+ CD4 and CD8 T cells in draining LN (n=3–7/group, (male), representative of 3 independent experiments (repeated in females)) (F). (G) Footpads of 12 week old OT-I mice were immunized with OVA as described in Figure 1B. Flow cytometric analysis of CD25+ IFN-γ+ OT-I CD8 T cell in draining LN (n=5/group (female), representative of 2 independent experiments). (H-J) Naïve CD8 T cells from 10–12 week old female or male OT-I mice were sorted and co-cultured with BMDC in the presence or absence of SIINFEKL (OVAp) and IL-12. Illustration of BMDC and CD8 T cell co-culture experimental model (H). Flow cytometric analysis of CD25+ IFN-γ+ OT-I CD8 T cells after 16 hours of co-culture (left) Flow cytometric analysis of mean florescent intensity (MFI) of CD25 and IFN-γ of CD8+ cells after co-culture (right, n=3–6/group (female), representative of four independent experiments (repeated in males)) (I). Sorted naïve OT-I CD8 T cells (single cells/live/CD45+/CD3ε+ CD8α+) were labeled with Cell Trace Violet (Violet). Cell proliferation was measured with Violet dilution after 48 hours of OVAp activation (n=6/group, female). Data are represented as mean ± SEM. 1-way ANOVA (C, D, and F), 2-way ANOVA (E and I) Student’s t-test (rest), *p<0.05, **p<0.01, ***p<0.001 vs Control group, †p<0.05 vs both Control and DEX group.

Figure 2.. Perinatal GC exposure led to enhanced tumor growth and reduced anti-tumor CD8 T cell response in adulthood.

(A) B16-F10 melanoma growth in 12 week-old syngeneic B6 mice (left, n=7–8/group (male), representative of 3 independent experiments (repeated in females)) or allogenic Balb/c mice (right, n=4/group (male), representative of 2 independent experiments including females). (B) Survival of mice after B16-F10 tumor implantation (n=9–10/group (male)). (C) B16-F10 melanoma was harvested from 13 week old B6 mice after 7 days of tumor implantation, digested with collagenase IV, and analyzed with flow cytometry (after gating on single cells/live/CD45+) (n=6–10/group (female and male). (D) YUMMER1.7 melanoma growth in 11–12 week old B6 mice (n=8/group (male), representative of 2 independent experiments including females). (E and F) 10 week old B6 mice or OT-I mice were implanted with EG.7-OVA lymphoma, and the tumor growth was monitored. Tumor score in B6 mice (n=7 (female)) and OT-I mice (n=4–6/group (female)) (left) and tumor growth (right) (E). Representative picture of OT-I mice after 4 weeks of tumor implantation (F). (G) E.G7-OVA tumor was implanted to 8 week old wild-type mice, and OT-I CD8 T cell were sorted from 8 week old OT-I mice and adoptively transferred to the tumor-bearing mice. Experimental scheme with OT-I transfer and activation (top) and E.G7-OVA growth (bottom). Data are represented as mean ± SEM. 2-way ANOVA (A, C, D, E (right), G), Log-rank Mantel-Cox test (B and E (left)), *p<0.05, **p<0.01, ***p<0.001 vs Control group.

Figure 3.. Perinatal GC exposure elicited decreased anti-bacterial CD8 T cell function and increased bacterial burden.

(A and B) 16 week old B6 mice were infected with 1 × 10⁵ colony-forming unit (CFU) of LM-OVA. OVA-specific CD8 T cells in spleen of mice after 7 days post infection (n=6–7/group (female)) (A). Listeria burden in liver 7 days after LM-OVA infection (n=13/group (female)) (B). (C) 18 week old CD45.2+ B6 mice were infected with LM-OVA. On day 7 after infection, killing of antigen-loaded splenocytes was analyzed by flow cytometry (see also Figure S4A, n=5/group (female). (D-F) OT-I CD8 T cells were sorted from 12 week old OT-I mice and labeled with Violet, adoptively transferred to wild-type mice and then infected with LM-OVA. Flow cytometric analysis of OT-I cell proliferation in blood (after gating on single cell/live/CD45+) (n=5/group (male) in each time points) (D). Listeria burden in liver 3 days after LM-OVA infection (n=5–6/group (female)) (E). Flow cytometric analysis (after gating on single cells/live/CD45+/CD3ε+ CD8α+) of IFN-γ+ cells among Violet+ OT-I CD8 T cells (left, F), and Granzyme B expression in Violet+ OT-I CD8 T cells after 3 days post infection (n=6/group (female)) (right, F). Data are represented as median ± 95% confidence interval (B and E) or mean ± SEM (others). Mann Whitney U-test (B and E), Student’s t-test (A, C, and F), 2-way ANOVA (D), *p<0.05, **p<0.01, ***p<0.001 vs Control group.

Figure 4.. Perinatal GC exposure decreased systemic CORT level, and inhibition of GR signaling reduced CD8 T cell function.

(A) Corticosterone level in 16 week old B6 mice (n=14–19/group (female and male), ZT=3 (10 am)). (B) Corticosterone level in B6 mice after 5 days of B16-F10 melanoma implantation (n=5–7/group (male), left, ZT=4), after 7 days of LM-OVA (n=4/group (female), middle, ZT=4), and after 30 minutes of restraint stress (n=7/group (male and female), ZT=6). (C) Flow cytometric analysis of CD44+ IFN-γ+ OT-I CD8 T cells with or without RU486 (1 μg/ml) in BMDC and OT-I co-culture system described in Figure 1H (n=3/group (female), representative of 3 independent experiments including males). (D and E) MTY (800 μg/ml) was treated in drinking water during footpad immunization with LPS+IFA+OVA (−7 to +5 days of immunization) in mice, as described in Figure 1B. Flow cytometric analysis of IFN-γ+ CD8 T cells in the LN (16 week old Balb/c mice, n=4–7/group (male)) (D), and CD44+ IFN-γ+ OT-I CD8 T cells in the LN (16 week old OT-I mice, n=4–6/group (male)) (E). (F) 12 week-old Wild-type (WT;Nr3c1^fl/fl) mice or KO (CD4-Cre Nr3c1^fl/fl) mice were immunized with LPS+IFA+OVA. CD44+ IFN-γ+ CD8 T cells in LN were analyzed with flow cytometry (n=5–15/group (female and male)). (G and H) Bone marrow cells from 10 week old OT-I mice were transferred to 10 week old CD45.1+ wild-type mice. Footpads of recipient mice were immunized. Illustration of bone marrow transplantation experiment (G). Flow cytometric analysis of CD44+ IFN-γ+ CD8 T cells in LN (n=3–5/group (female)) (H). Data are represented as mean ± SEM. 1-way ANOVA (D and F) 2-way ANOVA (C), Student’s t-test (rest), *p<0.05, **p<0.01, ***p<0.001 vs Control (Vehicle, DMSO, WT Control) group.

Figure 5.. Reduction of GR signaling led to insufficient CD8 T cell response via reduction in survival and activation signaling.

(A and B) Footpads of 13 week old WT (Nr3c1^fl/fl) mice and KO (CD4-Cre Nr3c1^fl/fl) mice were immunized and analyzed (described in Figure 1B). Flow cytometric analysis of dead CD8 T cells in LNs (n=6–8/group (female and male)) (A), and the expression of Bcl2 and CD69 in CD8 T cell (n=6–8/group (female and male)) (B). (C and D) Wild-type BMDC and naïve CD8 T cells from 14 week old WT and KO mice were co-cultured in the presence or absence of anti-CD3ε antibody and IL-12. Flow cytometric analysis of CD25+ IFN-γ+ CD8 T cells (n=3/group (female), representative of 3 independent experiments including males) (C). Flow cytometric analysis of expression of proteins in CD8 T cells after activation with anti-CD3ε antibody (n=6/group (female), representative of 2 independent experiments) (D). (E-G) CD8 T cells were sorted (single cells/live/CD45+/CD3ε+/CD8α+) from LNs of 12 week old male B6 mice after 5 days of footpad immunization. mRNA from CD8 T cells was isolated and analyzed with RNA-seq. Differentially-regulated top canonical pathways with IPA analysis (E). Representative transcripts per millions (TPM) (n=3/group) (F and G). (H) Wild-type BMDC and naïve OT-I cells sorted from 16 week old OT-I mice were co-cultured as described in Figure 1G. Flow cytometric analysis after 3–6 hours of SIINFEKL (OVAp) treatment with or without IL-12 (n=3/group (male), representative of 2 independent experiments including females). Data are represented as mean ± SEM. 2-Way ANOVA (C and H), Student’s t-test (rest), *p<0.05, **p<0.01, ***p<0.001 vs Control or wild-type (WT) group.

Figure 6.. Perinatal GC exposure persistently changed epigenomic state of naïve CD8 T cells via alteration of T-bet.

(A) Naïve CD8+ T cells were sorted (with gating on single cell/live/CD45+/CD3ε/CD8α/CD62L+ CD44-) from spleens and inguinal LNs of 12 week old OT-I mice with or without perinatal DEX exposure. mRNA from naïve CD8 T cells was isolated and analyzed with RNA-seq (n=3/group (female)). Differentially regulated representative genes are illustrated with volcano plot. (B-D) Naïve CD8+ T cells were sorted from spleens and inguinal LNs of 12 week old OT-I mice, and epigenomic state was analyzed with ATAC-seq (n=3/group (female)). Illustration of motif enrichment analysis of ATAC-seq peak (B). Accessibility of Ifng locus in different animals, aligned with T-bet-binding sites (C). Accessibility of Tbx21 locus in different animals, aligned with GR-binding sites (D).

Figure 7.. Perinatal GC and stress altered the HPA axis activity.

(A-C) Brain of 14 week old B6 mice was collected, sectioned and stained for GR and MR. Number of MR puncta of GR+ nucleus in dentate gyrus, CA3, CA1 region of hippocampus (A) and paraventricular nucleus of hypothalamus (B) (n=3/group (female), representative of 2 independent experiments). Representative image of dentate gyrus MR and GR expression (Scale bar: 30 μm, C). (D) CORT was measured in the morning of age-matched 9–20 week old B6 mice with various perinatal stressors such as prenatal restraint (3 hours/day during E12.5-E17.5, n=3 (female)), or perinatal cold exposure (12 hours/day during E13.5 to PND7, n=5 (female)) (left, D), prenatal cold exposure (6 hours/day during E14.5-PND0, n=6–10/group (female and male)) (middle, D), and postnatal poly (I:C) (single injection to pups on PND3, n=10/group (female)) (right, D). (E and F) CORT was measured in 8–20 week old age-matched B6 mice with various perinatal treatment regime. CORT level in adult the mice with mid-pregnancy DEX exposure (E7.5-E14.5, n=5/group (female)) (left, E), with late-pregnancy DEX exposure (E14.5-PND1, n=8/group (male) (middle, E), and with postnatal DEX exposure (PND0-PND14, n=5/group (male)) (right, E). CORT level in the adult mice received single injection of vehicle or DEX (0.5 mg/kg) on PND2 (n=4/group (female)) (F). (G) Flow cytometric analysis of CD8+ T cells upon footpad immunization described in Figure 1B (left, n=4/group (14 week B6 male)) and in BMDC/CD8 co-culture system as depicted in Figure 1H (right, n=3–6/group (13 week B6 female)). (H) Flow cytometric analysis of CD8+ T cells upon footpad immunization of 8–12 week old B6 mice, as described in Figure 1B. (I) Working model of long-term programming of CD8 T cell immunity by perinatal glucocorticoids. Data are represented as mean ± SEM. 2-way ANOVA (G (right) and H), Student’s t-test (rest), *p<0.05, **p<0.01, ***p<0.001 vs Control or saline group unless otherwise stated.