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Sertoli cells (SCs) are immune privileged cells found in the testis that function to immunologically protect maturing germ cells from immune destruction. This immune protection is due to the blood-testis-barrier, which prevents infiltration of cytotoxic immune cells and antibodies, and SC production of immunomodulatory factors, that favor a tolerogenic environment. The ability of SCs to create an immune privileged environment has led to the exploration of their potential use in the treatment of various diseases. SCs have been utilized to create a tolerogenic ectopic microenvironment, to protect co-grafted cells, and to deliver therapeutic proteins through gene therapy. To date, numerous studies have reported the potential use of SCs for the treatment of diabetes, neurodegenerative disorders, and restoration of spermatogenesis. Additionally, SCs have been investigated as a delivery vehicle for therapeutic products to treat other diseases like Laron syndrome, muscular dystrophy, and infections. This review will provide an overview of these therapeutic applications.

Patients with uncontrolled diabetes have an increased risk of developing cancer, therefore controlling their blood glucose and insulin levels is imperative. An effective treatment to accomplish this is transplantation of insulin-producing pancreatic islets. However, these patients must take chronic immunosuppressants to prevent graft rejection, which also increases cancer risk. An alternative to traditional transplantation is to transduce immunoregulatory cells such as Sertoli cells (SC), which survive as grafts without immune suppressants, to express insulin. We have previously transduced a mouse SC tumor line (MSC-1) to express human insulin (MSC1-HI), demonstrated that they survive (at 70-75%) when grafted into diabetic mice after 50 days, and confirmed their sustained expression of human insulin. Since complement is an important part of immune rejection, we hypothesize that one mechanism SC use to survive is inhibition of complement by expression of complement inhibitory proteins (CIP). We confirmed that primary mouse SC (pSC), MSC-1 cells, and MSC1-HI cells survive human complement activation in vitro while controls are killed, indicating that SC must be inhibiting complement, and microarray analyses of pSC and MSC-1 cells identified gene expression of 14 different CIP. We immunostained MSC-1 and MSC1-HI grafts to study deposition of C9, the lytic pore component of the complement membrane attack complex. In MSC1-HI grafts, C9 increased from about 10 positive cells/mm2 graft to roughly 40 positive cells/mm2 graft, and a dramatic increase in C9 deposition was observed on MSC-1 grafts—from about 4 positive cells/mm2 to well over 350 positive cells/mm2. Furthermore, we found that MSC1-HI grafts were significantly larger at day 50 than MSC-1 grafts. The MSC1-HI grafts also increased in size by almost 300% between days 20 and 50 post-transplantation as compared to MSC-1 grafts, which decreased in size by 64%. Thus, it appears that MSC-1 grafts are still rejecting while MSC1-HI grafts are growing, indicating some level of tumorigenesis. Indeed, insulin has been shown to be a growth factor for SC, hence we propose that increased expression of insulin in the MSC-1 cancer cell line is encouraging tumorigenesis in diabetic mice. Taken together, these data indicate that MSC1-HI cells undergo tumorigenesis and can survive and proliferate in spite of complement activation, which may be playing a role in MSC-1 graft reduction. Since complement can be a powerful anti-tumor and transplant rejection mechanism, understanding how these SC tumor cells evade complement killing has significance in both treating cancer and increasing grafted organ survival. As diabetes patients are more susceptible to tumors, further studying the relationship between insulin and tumorigenesis will increase knowledge to allow for more effective management of tumors. Citation Format: Rachel L. Washburn, Taylor Hibler, Jannette M. Dufour. A genetically engineered mouse Sertoli cell line survives chronic humoral immune factors when grafted into diabetic mice. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P044.

Advanced germ cells develop after central tolerance is established and express non-self antigen but are not destroyed by the immune system. They owe their survival in part to the physical and immunological barrier created by Sertoli cells (SC). The protective nature of SC has sparked interest in the field of transplant immunology as a way to prevent transplant rejection. Indeed, SC prolong the survival of various co-transplanted tissues. However, exactly how SC achieve this protection remains largely unexplored, as does their effectiveness in female recipients. Data from transplantation studies indicates that T regulatory cells (Tregs) are critical in graft survival. This study aims to determine if SC survive at comparable rates in males and females, and if SC survival is dependent on Tregs. Therefore, we transplanted SC from B6 mice into allogeneic BALB/c male and female immunosuppressive drugs. At day 20, all SC grafts survived in male recipients and at least 67% survived in female recipients. In both males and females, Tregs were detected in high numbers in surviving grafts. To further explore their role in SC graft survival, Tregs were depleted in male graft recipients. Analysis of SC grafts at day 20 showed that 57% of SC grafts survived and saw a return of Tregs, as detected through flow cytometry. Tregs remained depleted in the rejected grafts. Together, this data indicates that Tregs must be present for SC grafts to survive and that SC themselves may induce Tregs. To conclude, SC survive allotransplantation in males and females, possibly through the activation of Tregs. Supported by CH Foundation, NIAID

The acute cell-mediated immune response presents a significant barrier to xenotransplantation. Immune-privileged Sertoli cells (SC) can prolong the survival of co-transplanted cells including xenogeneic islets, hepatocytes, and neurons by protecting them from immune rejection. Additionally, SC survive as allo- and xenografts without the use of any immunosuppressive drugs suggesting elucidating the survival mechanism(s) of SC could be used to improve survival of xenografts. In this study, the survival and immune response generated toward neonatal pig SC (NPSC) or neonatal pig islets (NPI), nonimmune-privileged controls, was compared after xenotransplantation into naïve Lewis rats without immune suppression. The NPSC survived throughout the study, while NPI were rejected within 9 days. Analysis of the grafts revealed that macrophages and T cells were the main immune cells infiltrating the NPSC and NPI grafts. Further characterization of the T cells within the grafts indicated that the NPSC grafts contained significantly more cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) regulatory T cells (Tregs) at early time points than the NPI grafts. Additionally, the presence of increased amounts of interleukin 10 (IL-10) and transforming growth factor (TGF) β and decreased levels of tumor necrosis factor (TNF) α and apoptosis in the NPSC grafts compared to NPI grafts suggests the presence of regulatory immune cells in the NPSC grafts. The NPSC expressed several immunoregulatory factors such as TGFβ, thrombospondin-1 (THBS1), indoleamine-pyrrole 2,3-dioxygenase, and galectin-1, which could promote the recruitment of these regulatory immune cells to the NPSC grafts. In contrast, NPI grafts had fewer Tregs and increased apoptosis and inflammation (increased TNFα, decreased IL-10 and TGFβ) suggestive of cytotoxic immune cells that contribute to their early rejection. Collectively, our data suggest that a regulatory graft environment with regulatory immune cells including CD4 and CD8 Tregs in NPSC grafts could be attributed to the prolonged survival of the NPSC xenografts.

Sertoli cells (SC) are immunoregulatory testicular cells that protect and nurture germ cells, which develop after central tolerance is established and are classified as non-self by the immune system. This ability to protect germ cells makes SC a promising alternative to immunosuppressive therapy in transplantation; indeed, they have been co-transplanted with various tissues in animal models with significant success. It has been suggested that regulatory T cells (Tregs) play a role in SC graft survival and protection of germ cells. The goal of this study was to determine if SC promote graft survival through induction of Tregs. To test this, B6 SC were transplanted into BALB/c mice in absence of immunosuppressive drugs. 100% of SC grafts survived to day 20, while 100% of controls (nonimmune privileged cells) rejected by day 20. Analysis of the grafts through immunohistochemistry and flow cytometry revealed that SC grafts had significantly increased Tregs compared to rejecting control grafts. To determine if Tregs were critical for SC survival, B6 SC were transplanted into BALB/c mice depleted of Tregs. 100% of SC grafts without Tregs rejected within 20 days. Excitingly, 57% of SC grafts in Treg depleted mice survived. Flow cytometry revealed that all of the surviving grafts had Tregs, indicating that SC can induce Tregs from naïve T cells and that these Tregs are critical for SC graft survival. The presence of Tregs in surviving pig SC grafts was also shown in a xenogeneic model. Interestingly, the xenograft model showed a significant decrease in T effector cells, indicating that SC are both inducing Tregs and inhibiting effector cells. In summary, SC can survive as both allo- and xenografts without immunosuppression through the activation of Tregs.