Adaptive and Innate Immune Cells in Fetal Human Cytomegalovirus-Infected Brains

Abstract

Background: The understanding of the pathogenesis of cytomegalovirus (CMV)-induced fetal brain lesions is limited. We aimed to quantify adaptive and innate immune cells and CMV-infected cells in fetal brains with various degrees of brain damage.

Methods: In total, 26 archived embedded fetal brains were studied, of which 21 were CMV-infected and classified in severely affected (n = 13) and moderately affected (n = 8), and 5 were uninfected controls. The respective magnitude of infected cells, immune cells (CD8⁺, B cells, plasma cells, NK cells, and macrophages), and expression of immune checkpoint receptors (PD-1/PD-L1 and LAG-3) were measured by immunochemistry and quantified by quantitative imaging analysis.

Results: Quantities of CD8⁺, plasma cells, NK cells, macrophages, and HCMV⁺ cells and expression of PD-1/PD-L1 and LAG-3 were significantly higher in severely affected than in moderately affected brains (all p values < 0.05). A strong link between higher number of stained cells for HCMV/CD8 and PD-1 and severity of brain lesions was found by component analysis.

Conclusions: The higher expression of CD8, PD-1, and LAG-3 in severely affected brains could reflect immune exhaustion of cerebral T cells. These exhausted T cells could be ineffective in controlling viral multiplication itself, leading to more severe brain lesions. The study of the functionality of brain leucocytes ex vivo is needed to confirm this hypothesis.

Keywords: LAG-3; PD-1; cytomegalovirus; exhaustion; fetal brain; immune cells.

Figure 1

Immunohistochemistry results in one of the severely affected brain samples (case 7). DAB-chromogen was used, and slides were scanned with a NanoZoomer 2.0 HT Digital slide scanner. human cytomegalovirus (HCMV)-positive cells, CD8⁺ cells, CD20⁺ cells, plasma cells, NK cells (NKp46⁺, NKG2C⁺), and macrophages (CD68⁺ cells) are presented. Scale bar 50 µm

Figure 2

Repartition of HCMV-positive cells and immune cells (A) in one severely affected fetal brain sample (group A), successive cuts (case 8), and (B) in one moderately affected fetal brain sample (group B), successive cuts (case 17). Scale bar 35 mm.

Figure 3

Mean immunostaining densities of HCMV-positive cells and immune cells according to the severity of the brain lesions: severe (group A, N = 13), moderate (group B, N = 8), and controls (N = 5); * p < 0.05, ** p < 0.01, *** p <0.001. Kruskal–Wallis test was used with Dunn’s Multiple Comparison’s Test and Mann–Whitney test.

Figure 4

Detection, localization, and quantification of PD-1⁺ cells, PD-L1⁺ cells, LAG-3⁺ cells, and TIM-3⁺ cells in infected fetal brains. (A) Detection of PD-1, PDL-1, LAG-3, and TIM-3 immunostaining in one of the severely affected brains (case 7), scale bar 50 µm. (B) Localization of PD-1, PDL-1, LAG-3, and CD8 immunostaining in one of the severely affected brain samples and (C) in a moderate affected brain sample. (D) Mean immunostaining densities of PD-1 and LAG-3 according to the severity of the brain lesions: severe (group A), moderate (group B), and controls; ** p < 0.01, *** p < 0.001. Kruskal–Wallis test was used with Dunn’s Multiple Comparison’s Test and Mann–Whitney test.

Figure 5

Principal component analysis of CD8, or NKp46, or CD20 and viral multiplication (HCMV) and PD-1 in brain lesions according to cerebral severity (A (severe)/B (moderate)/controls). Severe cases are represented by red dots, moderate cases by yellow dots, and controls by blue dots.