High-dimensional multiomics reveals perturbations to IL-6/IL-6R axis and RUNX3 in CD4+ T cells during third trimester pregnancy

1. Problem Statement

CD4+ T cells are critical for maintaining maternal-fetal tolerance and regulating immune responses during pregnancy. However, the specific molecular adaptations of CD4+ T cells in the third trimester—a period associated with the highest risk of severe infection outcomes and obstetric complications (e.g., preeclampsia, fetal growth restriction)—remain poorly characterized. While imbalances in T-cell subsets (Th1/Th2/Th17/Treg) are hypothesized to drive complications, the precise surface proteomic and transcriptomic changes occurring in peripheral blood versus the decidual tissue (maternal-fetal interface) are not fully understood. This study aims to map these high-dimensional changes to identify mechanistic insights into pregnancy adaptation and potential targets for treating pregnancy complications.

2. Methodology

The study employed a high-dimensional multiomics approach comparing CD4+ T cells from three groups:

• Non-pregnant women (peripheral blood).
• Term-pregnant women (>37 weeks gestation) (peripheral blood).
• Term-pregnant women (decidual tissue from the same cohort).

Key Techniques:

• Surface Protein Screening (Flow Cytometry): A "LegendScreen" approach was used to screen >350 surface proteins on CD4+ T cell subsets (Naïve, Central Memory [TCM], Effector Memory [TEM], and Regulatory [Treg]) in both blood and decidua.
• Single-Cell RNA Sequencing (scRNA-seq) with CITE-seq: >130 CITE-seq barcoded antibodies were used alongside whole transcriptome sequencing to correlate surface protein expression with gene expression profiles.
• Cytokine/Receptor Quantification: Plasma concentrations of IL-6 and soluble IL-6 receptor (sIL-6R) were measured using LEGENDplex and ELISA.
• Bioinformatics: Dimensionality reduction (UMAP), differential gene expression (DEG) analysis, and Gene Set Enrichment Analysis (GSEA) were performed using Seurat and clusterProfiler.

3. Key Contributions

• Comprehensive Multiomics Atlas: Generated the first high-resolution map linking surface proteome and transcriptome of CD4+ T cells specifically in the third trimester of human pregnancy across blood and decidual compartments.
• Identification of the IL-6/IL-6R Axis Perturbation: Uncovered a paradoxical state where IL-6 signaling is enhanced (via soluble receptors and plasma cytokines) while surface IL-6R expression is downregulated on T cells.
• Discovery of RUNX3 in Decidual Tissue-Residency: Identified the transcription factor RUNX3 as a key marker for CD69+ tissue-resident-like CD4+ T cells in the decidua, linking it to differentiation and residency.
• Subset-Specific Adaptations: Detailed how specific T cell subsets (Naïve, TCM, TEM) adapt differently to the pregnant state, particularly regarding migration receptors (CCR5, CX3CR1) and activation markers.

4. Key Results

A. Surface Protein Adaptations

• Blood vs. Non-Pregnant:
  • Downregulation: CD9 and IL-6R (CD126) were decreased across Naïve, TCM, and TEM subsets. CD9 reduction may be linked to decreased platelet counts (lymphocyte-platelet conjugates).
  • Upregulation: CD85d (ILT4) increased in TCM; CD100, CD200R, and CD272 (BTLA) increased in TEM and Treg subsets.
• Decidua vs. Blood:
  • Migration Receptors: CCR5 and CX3CR1 showed the highest increase in Naïve, TCM, and Treg subsets in the decidua compared to blood. In the TEM subset, these receptors were already high in blood and remained consistent in the decidua.
  • Activation/Residency: CD85d (ILT4) was universally increased on decidual CD4+ T cells across all subsets, suggesting activation by fetal trophoblast-derived semaphorin 4A.

B. Transcriptomic and Signaling Perturbations

• IL-6/IL-6R Axis:
  • Protein Level: Surface IL-6R (CD126) was lower on Naïve and TCM cells in pregnant blood.
  • Transcriptomic Level: scRNA-seq of Naïve cells revealed enrichment in the IL-6/JAK/STAT3 and IL-2/STAT5 signaling pathways.
  • Plasma Level: Both IL-6 and sIL-6R concentrations were significantly elevated in pregnant plasma.
  • Interpretation: The study suggests a negative feedback loop or receptor shedding mechanism where high soluble IL-6R drives signaling despite low surface receptor density.
• TCM and Estrogen Response:
  • TCM cells in pregnancy showed enrichment for Estrogen Response (Early and Late) and TNF signaling.
  • Upregulation of anti-apoptotic genes (e.g., BCL2, BCL3) suggests enhanced survival mechanisms for CD4+ T cells during pregnancy.
• RUNX3 and Decidual Residency:
  • Decidual CD4+ T cells displayed an activated transcriptome (upregulation of GZMK, PRF1, CD69, IFNGR1).
  • RUNX3 expression was significantly higher in the CD69+ tissue-resident-like subset of decidual CD4+ T cells compared to CD69- cells. This subset also showed high CD95 (differentiation) and low CD62L (central memory), indicating a differentiated, tissue-resident phenotype.

C. Validation

• Flow cytometry validated transcriptomic findings, confirming lower CD62L on TCM and increased RUNX3 mean fluorescence intensity (MFI) in pregnant TCM cells.
• Discrepancies were noted for CD52 (high in both groups despite transcriptomic variation) and Granzyme K (higher in blood than decidua, contrary to transcriptomic trends), highlighting the necessity of multi-modal validation.

5. Significance and Implications

• Mechanistic Insight into Pregnancy Complications: The identified perturbations in the IL-6 axis and RUNX3-mediated residency provide potential mechanistic explanations for placental dysfunction. Dysregulation of these pathways could contribute to preeclampsia, fetal growth restriction, and stillbirth.
• Antiviral Immunity: Understanding how CD4+ T cells adapt (e.g., altered migration via CCR5/CX3CR1 and survival via BCL2) is crucial for predicting susceptibility to viral infections (like Influenza or SARS-CoV-2) during the third trimester.
• Therapeutic Targets: The study identifies specific molecular features (e.g., the IL-6/sIL-6R balance and RUNX3 expression) that could serve as biomarkers for pregnancy complications or targets for immune modulation therapies to improve pregnancy outcomes.
• Limitations: The study is observational with a relatively small sample size (n=58 pregnant, n=56 non-pregnant, n=14 decidua), and confounders like gravidity, parity, and fetal sex were not fully accounted for. Future studies are needed to validate these findings across different gestational stages and larger cohorts.