Soluble Forms of Immune Checkpoints and Their Ligands as Potential Biomarkers in the Diagnosis of Recurrent Pregnancy Loss-A Preliminary Study

Abstract

Immune checkpoints (ICPs) serve as regulatory switches on immune-competent cells. Soluble ICPs consist of fragments derived from ICP molecules typically located on cell membranes. Research has demonstrated that they perform similar functions to their membrane-bound counterparts but are directly present in the bloodstream. Effective control of the maternal immune system is vital for a successful pregnancy due to genetic differences between the mother and fetus. Abnormalities in the immune response are widely acknowledged as the primary cause of spontaneous abortions. In our research, we introduce a novel approach to understanding the immune-mediated mechanisms underlying recurrent miscarriages and explore new possibilities for diagnosing and preventing pregnancy loss. The female participants in the study were divided into three groups: RSA (recurrent spontaneous abortion), pregnant, and non-pregnant women. The analysis of soluble forms of immune checkpoints and their ligands in the serum of the study groups was conducted using the Luminex method Statistically significant differences in the concentrations of (ICPs) were observed between physiological pregnancies and the RSA group. Among patients with RSA, we noted reduced concentrations of sGalectin-9, sTIM-3, and sCD155, along with elevated concentrations of LAG-3, sCD80, and sCD86 ICPs, in comparison to physiological pregnancies. Our study indicates that sGalectin-9, TIM-3, sLAG-3, sCD80, sCD86, sVISTA, sNectin-2, and sCD155 could potentially serve as biological markers of a healthy, physiological pregnancy. These findings suggest that changes in the concentrations of soluble immune checkpoints may have the potential to act as markers for early pregnancy loss.

Keywords: CTLA-4; PD-1; RSA; TIGIT; TIM-3; VISTA; immune checkpoints; miscarriage; pregnancy loss; recurrent spontaneous abortion; soluble immune checkpoints.

Figure 1

Relationship between antigen-presenting cell (APC), lymphocyte T, and trophoblast cells regulated by secreted immune checkpoints. The figure illustrates the intricate interplay among antigen-presenting cells (APC), T lymphocytes, and trophoblast cells, highlighting the impact of secreted immune checkpoints. The secretion of soluble immune checkpoints, including sPD-1, sCD80/86, sGal-9, sCD112, sCD155, etc., is depicted. Elevated soluble factors may lead to the T cell inactivation and downregulation of trophoblast antigen presentation by APC cells. The interaction of Gal-9 (Galectin-9) and PtdSer (phosphatidylserine) is crucial during implantation process. The figure is adapted from the work of Zych et al. (2021) [8], exploring differences in immune checkpoint expression (TIM-3 and PD-1) on T cells in women with RSA.

Figure 2

Concentrations of secretory molecules controlling the immune system (ICPs) and their ligands, (A) sCTLA-4, (B) sCD28, (C) sCD86, (D) sCD80 in the sera of studied groups of women: Group of RSA women (n = 18), group of pregnant women (RSA) (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points, with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05.

Figure 2

Concentrations of secretory molecules controlling the immune system (ICPs) and their ligands, (A) sCTLA-4, (B) sCD28, (C) sCD86, (D) sCD80 in the sera of studied groups of women: Group of RSA women (n = 18), group of pregnant women (RSA) (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points, with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05.

Figure 3

Concentrations of secretory molecules controlling the immune system: (A) sPD-1, (B) sPD-L1, (C) sPD-L2 in the sera of studied groups: Group of women with miscarriages (RSA) (n = 18), group of pregnant women (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points, with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test.

Figure 4

Concentrations of secretory molecules controlling the immune system, (A) sVISTA, (B) sHVEM in the sera of studied groups: Group of women with miscarriages (RSA) (n = 18), group of pregnant women (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points, with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05.

Figure 5

Concentrations of secretory ligands. (A) sNectin2, (B) sCD155 in the sera of studied groups: Group of women with miscarriages (RSA) (n = 18), group of pregnant women (n = 16), group of non-pregnant women (n = 9). Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05.

Figure 6

Concentrations of secretory molecules controlling the immune system. (A) sTIM-3, (B) sLAG-3, in the sera of studied groups: Group of women with miscarriages (RSA) (n = 18), group of pregnant women (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05.

Figure 7

Soluble Galectin-9 concentration in studied groups: Group of women with miscarriages—RSA (n = 18), group of pregnant women (n = 16), group of non-pregnant women (n = 9). Results are presented as individual data points with the mean value indicated as a line. Significance was calculated using Student’s t-test or Mann–Whitney U test, * p < 0.05. The statistically significant differences marked with lines.