To characterize the fetal immunologic landscape in pregnancies complicated by maternal SARS-CoV-2 infection, we performed droplet-based single-cell RNA sequencing (scRNAseq) of CBMC from infants born to mothers with SARS-CoV-2 infection during pregnancy (cases) and infants born to mothers without SARS-CoV-2 infection (controls). CBMCs from three cases and three controls were obtained from our biorepository25. None of the three infants in this study born to mothers with SARS-CoV-2 were positive for SARS-CoV-2 postnatally, had detectable SARS-CoV-2 mRNA in placenta or developed any neonatal morbidity. All mothers with COVID19 in the third trimester were classified as having mild disease without respiratory support26. Infants born to mothers negative for SARS-CoV-2 and asymptomatic (universal screening at admission for labor) during the same epoch served as controls. Maternal comorbidities were matched between cases and controls as feasible. Table 1 displays demographic and clinical data from the cases and controls.
CBMCs were processed on the 10X Genomics Single-Cell Immune platform (see Methods). After quality control and doublet removal, we included 14,748 cells with high quality single-cell transcriptomes from cases and 11,222 cells from controls in our dataset. (See quality control metrics in Supplementary Fig. 1A-B). The cellular population composition was visualized using uniform manifold approximation and projection (UMAP, Fig. 1A), and cell types were inferred by cluster-specific canonical marker genes (Fig. 1B-C). We did not observe any differences in cell cluster composition between cases and controls (Supplementary Fig. 1C).
To explore transcriptional signatures in fetal immune cells associated with maternal SARS-CoV-2 infection, we performed differential gene expression (DGE) analysis within cell types comparing cases and controls. Genes with a false discovery rate (FDR) < 5% were considered statistically significant. We identified hundreds of genes across nearly all cell types with altered expression (Fig. 2A). We used gene ontology (GO) analysis to broadly classify genes significantly disrupted by maternal SARS-CoV-2 infection based on DGE (Supplemental Table 1).
CD14 + monocytes were grouped into 5 clusters and CD16 + monocytes were grouped into one cluster (Fig. 2B). CD14 + sub-populations demonstrated variable expression of inflammatory genes, including ACSL1, ADGRE2, CD300E and PADI4, which aligns with prior single cell analysis showing monocyte diversity27. Consistent with data from adult COVID-19 patients28,29, we found that CD14 + monocytes from cases demonstrated increased expression of ISG (Fig. 2C) and concomitant IFNAR2 downregulation (Supplemental Fig. 2a), which could reflect exposure to interferon prenatally30. GO analysis of DGE in CD14 + monocytes demonstrated enrichment of genes associated with antigen presentation and viral translational termination and reinitiation (Fig. 2D). Cord blood (CB) CD14 + monocytes from cases also showed upregulation of major histocompatibility class (MHC) I and II genes suggesting activation in response to interferon signaling31. Furthermore, CD14 + monocytes from cases showed upregulation of TLR receptor transcripts (TLR2, TLR4 and TLR5) paired with upregulation of FOS and downregulation of transcriptional inhibitors of NFKB (NFKBIA and NFKBIE), all of which are associated with increased NFKB activation and cytokine production32 (Supplemental Fig. 2A). Of note, CD14 + monocytes from cases had decreased expression of autophagy (ATG14, ATG2A, ATG3) and endoplasmic reticulum stress (XBP1, HSPA5) genes, which may contribute to a defect in macrophage differentiation33 (Supplemental Fig. 2A).
Similar to CD14 + monocytes, we identified induction of ISG in non-classical CB monocytes (CD16+) (Fig. 2C). In contrast to CD14 + monocytes, we found that there was decreased expression of cell adhesion genes (including PLAUR and THBS1), attenuation of immune activation signaling pathways genes (FOS, FOSB, MAP3K8, STAT6, and FCER1G), and decreased expression of inflammatory molecules like resistin (RETN) (Supplemental Fig. 2B). Together, these results suggest induction of ISG in monocytes from cases compared to controls and differences in transcriptional changes in classical and non-classical monocytes that might suggest preferential activation of classical monocytes in cases compared to controls.
We captured the transcriptomes of both plasmacytoid and conventional dendritic cells (pDC and cDC, respectively) in CB. In adults infected with SARS-CoV-2, both types of DCs are functionally impaired, and there is an increased ratio of cDCs to pDCs in severe patients34. In our study, CB cDC from cases showed increased expression of ISG like IFITM3 and APOBEC3A (Fig. 2E). Transcription factor zinc finger E box–binding homeobox 2 (Zeb2) plays a crucial role in promoting cDC and pDC development by downregulating Inhibitor of DNA binding protein 2 (ID2)35,36. ZEB2 was increased in cDC and ID2 was decreased in pDC from CB of cases, which might be evidence of a shift towards pDC in CB from infants exposed to SARS-COV-2 in utero (Fig. 2E). Fetal cDC from cases showed a transcriptional profile suggestive of innate immune activation including increased expression of PIK3CB, which is downstream of TLR5 and TLR737, as well as increased transcription of CCL5, which can be upregulated after TLR3 stimulation (Fig. 2E)38. Evidence of impaired cDC maturation was suggested by upregulation of ID1, which antagonizes dendritic cell differentiation and antitumor immunity in mice39, as well as increased MAFB transcription, which suppresses cDC maturation40. cDCs from cases also demonstrated decreased expression of FOSB and many MHC II genes41. pDCs in cases also showed markers of immune activation, including upregulation of RELB, which promotes DC activation through RelB-p50 dimer42, upregulation of MHC Class I and Class II genes, and UPR activation, as shown by increased transcription of XBP143 (Supplemental Fig. 2C). Together, these transcriptional findings could be consistent with activation of pDC over cDC in the CB of cases, potentially through activation of TLRs.
In adults, SARS-CoV-2 infection is associated with fewer blood NK cells but a higher activation state in circulating NK cells44. We identified two clusters of cord blood NK cells. One population of NK cells (cluster 1) expressed higher levels of GZMB, while the second population of NK cells (cluster 2) expressed IL7R and XCL1, suggesting that cluster 1 corresponded to CD56dim and cluster 2 corresponded to CD56bright NK cells, as NCAM1 (CD56) is technically not well captured in scRNAseq45. Similar to adult NK cells, CB NK cells from SARS-CoV-2-positive pregnancies showed signs of exposure to interferon, including induction of ISG genes like IFI6, IFIT2 and IRF9 (Fig. 2F)44,46,47 We identified increased transcription of CCL4, expression of cytotoxic genes including GNLY, GZMA, GZMB and GZMH, and increased transcription of IFNG, paired with decreased expression of NK inhibitory molecules (Fig. 2F)44,46,47. There were transcriptional changes associated with exhaustion, such as decreased expression of KLRG1 and SIGLEC748. DGE in NK cells between cases and controls were enriched for genes related to the interferon-alpha response, regulation of NK cell cytokine production, and viral transcription (Fig. 2G).
In adults with acute COVID19, there is a heterogeneous adaptive immune response in peripheral blood, including B-cell receptor and T-cell receptor arrangements specific to SARS-CoV-246. Given these findings, we evaluated whether maternal infection with SARS-CoV-2 had any effect on CB lymphocyte gene expression. We were able to identify three clusters of CB B-cells (Fig. 1A,B). We also identified three clusters of T-cells (Fig. 3A). Cluster 1 corresponded to CD8 + T-cells. Cluster 2 and 3 corresponded to helper T cells49. Increased expression of CCR7 in T-cell Cluster 2 suggests this cluster includes either naive T-cells or central memory T-Cells50 and increased CTSW and KLRB1 in Cluster 3 suggest this cluster includes effector and memory T-cells51,52.
In B cells from infants exposed to SARS-CoV-2 in utero, we identified 3 clusters of CB B-cells corresponding to non-plasma (Cluster 1 and 2) and plasma cells (Cluster 3) based on MZB1 expression46 (Supplementary Fig. 2D). In B-cells from infants born to mothers infected with SARS-CoV-2, we identified decreased markers of B-cell receptor activation in all clusters. Specifically, we found decreased transcription of NR4A1, CD69 and CD83 in all B-cells (Supplemental Fig. 2E). NR4A1 encodes Nur77, an orphan nuclear receptor, that is induced upon B-cell activation in peripheral blood in humans53. CD83 is expressed upon activation of B-cells, and activated T-cells induce CD83 on B-cells via CD40 engagement54. Concordant with decreased B-cell activation, we also found downregulation of CD6955, decreased expression AP-1 and NFAT genes56, and decreased expression of anti-apoptotic genes, including BCL2 and BCL2A157 (Supplemental Fig. 2E). Transcriptional changes suggestive of potential B-cell dysfunction, combined with decreased transplacental transmission of IgG against SARS-CoV-2 compared to IgG against other antigens6,58, might translate into potential impairments in antibody mediated immunity to SARS-CoV-2 in neonates born to mothers with COVID19.
In adults with COVID19, CD8 + T-cells show decreased cytotoxic potential and exhaustion driven by IL-659. Similar to adults, CB CD8 + T-cells from cases demonstrated transcriptional signatures suggestive of impaired cytotoxic activity, including decreased expression of GZMA60, and CD8 T-cell exhaustion, including downregulation of FOS (Fig. 3B)61. Furthermore, there was increased expression of genes associated with central memory T-cells, including KLRB152 and CCR762, that might be associated with fetal CD8 activation with SARS-CoV-2 (Fig. 3B). GO analysis of DGEs in CD8 + T-cells demonstrated enrichment for genes associated with T-cell tolerance, proliferation, and the response to interferon gamma (Supplemental Fig. 3A). In T-cell Cluster 2, we found increased expression of IL6-IL17 axis genes including RORA, ARID5A, RBPJ, and IL6ST in cases compared to controls (Supplemental Fig. 3B). IL6-IL17 axis has been implicated in mediating the neurodevelopmental effects of maternal immune activation in mice63.
T-cell antigen receptor (TCR) repertoire in T-cells reflects selection by self and foreign antigens. To investigate the repertoire of TCRs in CB from SARS-CoV-2-exposed pregnancies and controls, we performed single cell TCR sequencing. A total of 1,943 T-cells were analyzed, and T-cells with TCR information were well equally distributed between subject and T-cell populations (Supplemental Fig. 3C and 3D). Clonal expansion was significantly increased in T-cells from pregnancies complicated by maternal SARS-CoV-2 infection, with 40.4% of T-cells having > 5 clones in the cases, compared with 30.9% in the controls (Kolmogorov-Smirnov Test p value 2.2e-16) (Fig. 3D). The T-cell clonal expansion in CB from cases is consistent with results of T-cell repertoire analysis from adults infected with SARS-CoV-246.
Despite the novelty of scRNAseq analysis of CBMC, our study is exploratory and has several limitations. Importantly, the small number of samples limits the generalizability of our conclusions. However, few studies have evaluated CB immune populations by single-cell transcriptomics64,65, and our results illustrate an important and potentially underrecognized population in the COVID-19 pandemic that should be further studied. All cases included in this study were classified as mild maternal SARS-CoV-2 infection; more severe maternal infection could result in more dramatic or different fetal immune genomic signatures. Furthermore, the time from infection to delivery and cord blood collection likely affects the immune phenotype observed in cord blood. As the time of maternal infection and birth in our cohort fluctuates between 7 and 66 days, more pronounced findings could be found with samples with a more consistent timing between infection and collection. Lastly, all mothers affected with SARS-CoV-2 in our cohort had comorbidities including well-controlled thyroid dysfunction, obesity or gestational diabetes. Although we included mothers with similar comorbidities in the control population (except for gestational diabetes) and all these comorbidities were medically managed, it is possible that our results are influenced by the comorbidities of the mothers. However, thyroid disease, obesity or gestational diabetes in the mother have not been reported to trigger the transcriptional response patterns we observed in cases compared to controls66–69.
Despite these limitations, the present study identifies transcriptional changes suggestive of a fetal immune response after maternal infection with SARS-CoV-2 in the absence of vertical transmission and suggest potential trans-placental immune implications of maternal SARS-CoV-2 infection beyond vertical transmission. The source of signals promoting transcriptional changes in neonatal monocytes and other immune cells in the absence of vertical transmission is unknown. Ex-vivo studies have shown that transplacental transfer of IL1ꞵ, IL6 and TNF does not occur70. Type I interferons are increased in peripheral circulation of patients with mild COVID1928, but the ability of interferon to cross the human placenta is unclear71. Our results raise the possibility that pro-inflammatory signaling in the mother in response to SARS-CoV-2 might promote interferon signaling at the feto-maternal interface. Further experimental data needs to be collected to clarify how maternal infection with SARS-CoV-2 influences the fetal immune system. Given the extensive literature linking maternal immune dysregulation and abnormal fetal development in viral infections, this study raises important questions about untoward effects of maternal SARS-CoV-2 on the fetus, even in the absence of vertical transmission and highlights the need of further studies to better characterize the fetal immune response in pregnancies affected by SARS-CoV-2 infection.