T cell subsets in the human testis and their potential role in testicular germ cell tumors

Abstract

As an immune-privileged organ, the normal testis immune environment consists of macrophages, mast cells and few T cells, all having anti-inflammatory effects to protect developing germ cells from autoimmune attack. Recruitment of infiltrating immune cells is commonly observed in human testicular germ cell tumors (TGCT), i.e. seminoma, but the significance for disease progression and prognosis remains unknown. T cells represent the major component of tumor infiltrating lymphocytes (TIL) in TGCT. Potential roles of rarer subtypes, such as regulatory (Treg) and follicular helper T (Tfh) cells, being associated with tumor biology in other cancer entities, have not been investigated in TGCT. This study provides a detailed analysis of TIL focusing on Treg and Tfh in human testicular specimens characterized by hypospermatogenesis and lymphocytic infiltrates (ly) (n=12), pre-invasive germ cell neoplasia in situ (GCNIS -/+ly) (n=15, each), and seminoma (n=28) compared to normal spermatogenesis (n=10). Using immunohistochemistry (IHC), T cells were confirmed as most abundant TIL, with Treg and Tfh cells most frequently observed in seminomas. TIL from fresh human testicular tissue specimens (seminoma n=12; embryonal carcinoma (≥80%) n=6; mixed TGCT n=6) were analyzed by flow cytometry from different areas of tumor-bearing and contralateral (control) testes. Consistent with IHC, T cells were most abundant in seminomas. Treg and Tfh cells were most frequently detected in tumors, along with helper (CD4+) and cytotoxic (CD8+) T cells. For further in-depth analyses of T cell subsets and their signatures in human testis samples, data from scRNA-sequencing of normal testis (n=3; pooled data) and TGCT (n=4) was examined. Again, T cells were the most prominent TIL in TGCT. Secondary clustering showed that, in addition to CD8+ cytotoxic, proliferating (activated), CCR7+ (newly recruited), and other CD4+ T cells, Treg and Tfh were present with variable frequencies. Downstream analysis suggested several possible modes of TIL regulation: Treg cell recruitment in TGCT might be regulated by the interaction of NANOG with CXCR4; Treg functionality might be regulated by a cooperation between BATF and CCL5. In addition, the interaction between BCL6 and NANOG might control Tfh cell recruitment in TGCT, and the cooperation between CXCR3 and ICOS is likely to be important for their functionality. These in silico analytical findings are a fertile field for further experimental investigations. In summary, despite high inter-individual variation and sample heterogeneity, all study approaches showed a similar composition of immune cells in the human testis, with an overall increase of immune cells in TGCT compared to other pathological conditions and normal controls. In addition, it became clear that the predominance of resident macrophages in normal testes is shifted in favor of T cells as the main component of TIL in TGCT. Furthermore, the number of Treg and Tfh was significantly increased in TGCT compared to other pathological conditions and normal testis, possibly providing a tumor growth advantage. This study has described the complexity of TIL in TGCT and provides first indications that rarer T cell subtypes and their signaling molecules are likely to be of functional importance in the TGCT immune environment.