Phenotypic characterization of testicular immune cells expressing immune checkpoint molecules in wild‐type and pituitary adenylate cyclase‐activating polypeptide‐deficient mice

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a neuropeptide having several regulatory functions in the nervous system and in peripheral organs including those of the reproductive system. PACAP‐deficient male mice have several morphological, biochemical, behavioral defects and show disturbed signaling in spermatogenesis affecting fertility in PACAP KO mice. Reproductive functions such as fertility, mating, and maternal behaviors have been widely investigated, but no immune analyses are available regarding the testicular immune‐privileged environment in male PACAP‐deficient mice.


| INTRODUC TI ON
There are certain "immunologically privileged" sites in our body, where immune responses are naturally suppressed. The anterior chamber of the eye, central nervous system, and pregnant uterus are all considered physiological immune-privileged sites in humans. 1 The mammalian testis also represents an immune-privileged organ 2 maintained through the coordination of systemic immune tolerance, the local physical structure, and active local immunosuppression.
The local immune modulatory milieu has been intensively investigated, but the mechanisms underlying immune privilege are still not well understood. [2][3][4] The blood-testis barrier (BTB) is much more complex than other blood-tissue barriers, and its function relies on the complex interaction between the anatomical, physiological, and immunological barriers. 3 Besides the anatomical BTB, testicular somatic cells, including Sertoli cells, peritubular cells, and Leydig cells together with the local active immunosuppression, play vital roles in maintaining the immune-privileged environment. 2,4 The testis has most types of immune cells found in interstitial spaces, including macrophages, α/β and γ/δ T lymphocytes, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells (DCs), and mast cells. 5 These cells are important in the maintenance of the special testicular immune environment.
Both in mice and humans, binding of TIM-3 to its ligand Gal-9 leads to the apoptosis of Th1 and Th17 cells and induces active immunotolerance. 9,10 Known ligands of PD-1 include PD-L1, which can be found on several immune cells (resting T cells, B cells, dendritic cells, macrophages), in various tissues, like placenta, heart, and spleen and by Sertoli and peritubular cells in the testis of mice. 6,11 In contrast to that, PD-L2 expression is limited to dendritic cells and macrophages. 12 The PD-1/PD-L1 pathway is thought to be associated with the promotion of peripheral tolerance and subsequent prevention of immune-mediated tissue damage by favoring Th2 development and expansion. 13,14 Immune checkpoint regulators are thought to actively participate in the immune defense of infections, prevention of autoimmunity, immune reaction during transplantation, tumor immune evasion, and maternal immune tolerance during pregnancy. 6 Collaboration between these mechanisms is beneficial to healthy testicular function. However, the disturbance of this physiological status can lead to orchitis leading to male infertility, followed by impaired androgen synthesis and diminished spermatogenesis. 15 Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that belongs to the glucagon/VIP/secretin family. 16 It has major relevance in the nervous system and in several peripheral organs, including reproductive organs. PACAP is one of the peptides having several regulatory functions in reproduction, 17,18 and regulating germ cell development in the ovary and testis. 19,20 Pituitary adenylate cyclase-activating polypeptide-deficient (KO) mice display various abnormalities in several physiological and pathological processes, including reproductive functions. 17,18 Lack of PACAP in male mice leads to disturbed signaling in spermatogenesis, 20 which could be an important factor responsible for reduced fertility in PACAP KO mice. 21 PACAP is a known modulator of innate and adaptive immune responses. 22 The aim of our research was to investigate the role of Immune checkpoint pathways in the maintenance of immune regulation in testicular immune-privileged environment, especially in PACAPdeficient mice. We investigated the expression pattern of TIM-3, PD-1, and Gal-9 on different immune cell subsets in the testis of wild-type and PACAP KO mice.

| Animal model
Generation of PACAP-deficient mice on CD-1 background was described earlier. 23 Two-month-old wild-type (WT) and homozygous knockout (KO) female mice were maintained on a 12-hour light/dark cycle at 20-22°C and 40%-60% humidity and were fed with standard feed pellets and tap water. Animals were handled in accordance with an

| Cell isolation from the testis
Three-month-old adult male mice (n = 10 WT and n = 11 KO) were killed by cervical dislocation, and both testes were removed. To avoid selective cell death or selective loss of surface proteins, mechanical disaggregation rather than enzymatic digestion was used to process the testicular tissue for isolating testicular immune cells. Later, testicles were homogenized thoroughly with a syringe plunger, and single-cell suspensions were prepared using

F I G U R E 1
Gating technique used to detect immune cell populations in the testis. CD45+ leukocytes were gated using SSC and FL-5 (PerCP) parameters. Endothelial cells which may fall inside the lymphogate were excluded by CD45 staining. Lymphogate was created based on physical characteristics typical of lymphoid cells using forward and side scatter parameters. All further analyses of testicular immune cells were performed on CD45+ cells only, by combining the lymphogate and CD45+ cell gate

| Statistical analysis
Statistical analysis was performed using statistical software SPSS version 23 package. Statistical comparisons were made using Student's t test. Differences were considered significant if the P value was equal to or less than .05.

| Immunophenotypic analysis of testicular immune cells from wild-type and PACAP KO mice
We investigated the percentage of CD3+ T, CD4+ T, and CD8+ T cells; γ/δ T cells; Treg cells; and NK and NKT cells in the testis of wild-type and PACAP KO mice. We observed a significant increase in the CD3+ and CD8+ T-cell frequency ( Figure 3A,C), while the γ/δ T and Treg cells ( Figure 3D,E) significantly decreased in the testis of PACAP KO mice compared with the wild-type. The frequency of CD4+ T cells, and NK and NKT cells ( Figure 3B, F, and G) showed no difference between the investigated groups.

| TIM-3 and PD-1 expression by testicular immune cells from wild-type and PACAP KO mice
Flow cytometry was used to analyze the cell surface expression of TIM-3 by CD3+ T, CD4+ T, CD8+ T, γ/δ T, and NK cells in the testis of wild-type and PACAP KO mice.
We found that TIM-3 expression by CD4+ T and γ/δ T cells was significantly decreased in PACAP KO compared with wild-type mice

| Perforin expression by testicular immune cells from wild-type and PACAP KO mice
Flow cytometry was used to analyze the perforin expression by CD3+ T, CD8+ T, and NK cells in the testis of wild-type and PACAP KO mice. CD3+ and CD8+ T cells from PACAP KO mice showed significantly higher perforin expression compared with wild-type mice ( Figure 7A,B). No significant difference in the expression regarding perforin by NK cells was detected between the investigated groups ( Figure 7C).

| D ISCUSS I ON
The testis is a very complex organ with a unique structure and a large number of cell types. The mammalian testis consists of two distinct compartments: the seminiferous tubules responsible for spermatogenesis and the interstitial spaces between the tubules containing steroidogenesis performing cells. The interstitial spaces represent

are T cells, with CD8+ cells being predominant and CD4+ T cells are
rather infrequent. 5 In infertile patients with sperm autoimmunity, the number of lymphocytes is significantly higher, suggesting their role in testicular pathogenesis during inflammatory conditions. 25 The rat testis also contains immunoregulatory T cells, including Tregs, NKT, and γ/δ T cells. 25 Different experimental models have also shown the important role of macrophages in orchitis development in rats. 25,26 Several studies have shown the presence of PACAP and its receptors in human and mouse testis, 20,27,28 and it has been shown that PACAP can cross the BTB. 29  Tregs modulate the function of a variety of immune cells and are critical for maintaining self-tolerance, suppressing autoimmunity and preventing graft rejection, which has been reported in many animal models. 25 Nasr et al showed that antigen-specific Tregs promote transplantation tolerance to pancreatic islet allografts in testis. 33 We quantified other T-cell subsets, like Foxp3+ Treg cells and γ/δ reduced implantation was also observed in PACAP-deficient mice. 39 Based on these findings, PACAP affects reproduction at basically all levels. However, our study can further reveal another possible candidate to explain the abovementioned reduced fertility. Male seminal fluid is implicated in regulating the size and suppressive function of the Treg cell pool during the peri-implantation phase of early pregnancy leading to a state of active tolerance during embryo implantation to prevent the conceptus from maternal immune attack. 40,41 PACAP deficiency-related changes could modify the immunomodulatory effects of seminal fluid by altering either its cytokine signals or antigen content and also the male leukocyte priming during maternal implantation.
In summary, in our present study, we found that the increased