 and mucin domain-3 (TIM-3) receptor. The TIM-receptor family consists of three members in humans (TIM-1,-3,-4) and eight in mice (TIM-1-8). They are expressed by various immune cells, including T, NK, and antigen-presenting cells, and implicated in the regulation of pivotal immunological processes such as T cell proliferation, survival, and tissue inflammation [24,25]. The amount of data supports that TIM-3 is a potent regulator of both the adaptive and innate immune response, and negatively regulates T cell responses by inducing the apoptosis of Th1 cells [26]. To date, numerous studies investigated the interaction between Gal-9 and TIM-3 and found that the commitment of TIM-3 by Gal-9 may function as a regulator of Th1 immunity by abrogating Th1/Th17-driven immune responses, therefore modulate the Th1/Th2 cytokine balance [20]. A growing body of evidence demonstrates that galectins are widely expressed in the female reproductive system. Although it is well established that these lectins can control host-pathogen interactions, the function of the endometrium, the local immune response as well as implantation, placentation, and immune tolerance towards the fetus [27], their exact role in endometriosis is poorly examined. Previous literature data indicate that Gal-9 is mainly expressed by endometrial epithelial cells [28], and its production can be induced by tissue damage or pro-inflammatory cytokines (IFNγ, IL-1β, TNFα [29,30]). Furthermore, we found an overexpression and an elevated serum level of Gal-9 in endometriosis [31]. In this regard, we hypothesize, that the dysregulated TIM-3/Gal-9 immune checkpoint pathway has a crucial role in the immuno-pathogenesis of this disease.
Here we demonstrate that the distribution and the cell surface expression of the TIM-3 receptor and the Gal-9 ligand are significantly altered on different peripheral and peritoneal T and NK cell subsets of endometriosis-affected patients. Furthermore, our results indicate a persistent activation and a dysregulated TIM-3-dependent regulatory pathway in endometriosis, which may impair the local immune-surveillance mechanisms, could promote the survival of ectopic endometriotic implants and aid the development of endometriosis-associated reproductive failures.

Immunophenotypic Characterization of Peripheral Blood and Peritoneal Fluid Mononuclear Cells in Patients with Endometriosis and Non-endometriotic Women
As the exact immunophenotypic composition of the peripheral blood (PB) and peritoneal fluid (PF) immune cell populations in endometriosis were not yet characterized by our Research Team and previous literature data provided inconsistent results, our first aim was to determine the distribution of different T and NK cells in the peripheral blood mononuclear cell (PBMC) subsets of non-endometriotic (NE) control and endometriosis (E)-affected women and in the peritoneal fluid leukocytes (PTL) of the examined patients. Although all E-cases were confirmed by laparoscopy and their demographic data was evaluated in detail (Table 1), but the clinical classification was not done as the patients suffered from different forms and various extents of the disease. Unfortunately currently, neither the best-known rASRM nor the ENZIAN scoring system could accurately be used for the unified, comparative classification of all lesion types [32][33][34]. Moreover, the parallel use of both scoring systems for classification would be inappropriate as they refer to the different locations of lesions. Although the revised ENZIAN scoring may provide an excellent complement to the rASRM score for a morphological description of deep infiltrating endometriosis (DIE), however, this system is not in use in our clinical practice [35]. In the case of NE-cohort, under the EU-General Data Protection Regulation (EU-GDPR) and the Privacy, Informational, and Healthy data Act, with special regard to the confidentiality and privacy of voluntary non-remunerated blood donation, all personal and health-related data obtained about the donors during blood donation were processed anonymously, confidentially and securely and were not disclosed to the Research team for analysis. During analysis, the percentage of CD3+ T, CD4+ T, CD8+ T, Treg lymphocytes, as well as total CD56+ NK, CD56 dim NK, CD56 bright NK, and NKT-like cells were determined and compared based on the flow cytometric gating strategy indicated on Figure 1. During analysis, the percentage of CD3+ T, CD4+ T, CD8+ T, Treg lymphocytes, as well as total CD56+ NK, CD56 dim NK, CD56 bright NK, and NKT-like cells were determined and compared based on the flow cytometric gating strategy indicated on Figure 1. We found that in the E-PBMC samples, a significantly lower number of CD3+ T, CD4+ T, and Treg cells were detected; however, the frequency of total NK and CD56 dim NK cells was significantly higher in the affected group when compared to NE samples. As peritoneal fluid was also collected, we could compare the local and peripheral distribution of these subsets in endometriosis. In the E-PTL samples, we detected a significantly higher number of CD8+ T, CD56 bright NK, and NKT-like cells than in the periphery. In contrast, the frequency of CD4+ T and Treg cells were significantly lower in the E-PTL samples than in the E-PBMC (Table 2).  We found that in the E-PBMC samples, a significantly lower number of CD3+ T, CD4+ T, and Treg cells were detected; however, the frequency of total NK and CD56 dim NK cells was significantly higher in the affected group when compared to NE samples. As peritoneal fluid was also collected, we could compare the local and peripheral distribution of these subsets in endometriosis. In the E-PTL samples, we detected a significantly higher number of CD8+ T, CD56 bright NK, and NKT-like cells than in the periphery. In contrast, the frequency of CD4+ T and Treg cells were significantly lower in the E-PTL samples than in the E-PBMC (Table 2). Statistical comparisons were performed by Student's t-test between peripheral blood mononuclear cells of non-endometriotic women (NE-PBMC) vs. patients with endometriosis (E-PBMC), and between peripheral (E-PBMC) vs peritoneal leukocytes of endometriosis affected women (E-PTL). The calculated results were presented as the mean value ± standard deviation (mean ± SD) and the differences were considered significant when the p-values were ≤0.05. The percentage of lymphocyte subpopulations and their p-values indicating significant differences are marked in bold and "n.s." marks non-significant values. It is known that the level of FoxP3 expression by Treg cells is proportional to their suppressive capacity [36]. As literature data indicate that Treg dysfunction plays an important role in the development of endometriosis, we compared their relative FoxP3 expression in NE-PBMC, E-PBMC, and E-PTL cohorts. As indicated in Figure 2, the Mean Fluorescent Intensity (MFI) values of FoxP3 by Treg cells were significantly lower in the E-PBMC samples than that of the NE-PBMC specimens and showed the lowest MFI values when compared to the E-PTL Tregs of the evolved patients.
between peripheral (E-PBMC) vs peritoneal leukocytes of endometriosis affected women (E-PTL). The calculated results were presented as the mean value ± standard deviation (mean ± SD) and the differences were considered significant when the p-values were ≤0.05. The percentage of lymphocyte subpopulations and their p-values indicating significant differences are marked in bold, "n.s." marks non-significant values. It is known that the level of FoxP3 expression by Treg cells is proportional to their suppressive capacity [36]. As literature data indicate that Treg dysfunction plays an important role in the development of endometriosis, we compared their relative FoxP3 expression in NE-PBMC, E-PBMC, and E-PTL cohorts. As indicated in Figure 2, the Mean Fluorescent Intensity (MFI) values of FoxP3 by Treg cells were significantly lower in the E-PBMC samples than that of the NE-PBMC specimens and showed the lowest MFI values when compared to the E-PTL Tregs of the evolved patients.

Altered TIM-3 Expression by Peripheral Blood and Peritoneal Fluid Mononuclear Cell Subsets in Patients with Endometriosis and Non-Endometriotic Women
Previously, Popovici et al. [28] have found that the mRNA and protein expression of Gal-9 is exclusively increased at the mid-and late-secretory and decidual phases of normal endometrium. Furthermore, our recent study demonstrated a Gal-9 mRNA overexpression in ectopic endometriotic lesions and a significantly increased soluble Gal-9 level in the serum of endometriosis-affected patients compared to non-endometriotic controls [31]. Although these data pose a fundamental role of Gal-9 in endometriosis, the involvement of the TIM-3/Gal-9 regulatory pathway in the development of endometriosis-associated immunological abnormalities is not yet known. Therefore, our next goal was to characterize the cell surface expression of the TIM-3 immune-checkpoint receptor on different PBMC subsets of the NE group and in the E-PBMC and E-PTL samples of the endometriosis cohort.
When we compared the TIM-3 expression by different subsets of peripheral T cells, a significantly decreased labeling was observed on CD3+ T, CD4+ T, and CD8+ T cells, however, a significantly elevated positivity was found on FoxP3+ Treg cells of the examined patients when compared them