Specific immune cell and cytokine characteristics of human testicular germ cell neoplasia.
Which immune cells and cytokine profiles are characteristic for testicular germ cell neoplasia and what consequences does this have for the understanding of the related testicular immunopathology?



The unique immune environment of testicular germ cell neoplasia comprises B cells and dendritic cells as well as high transcript levels of IL-6 and other B cell supporting or T helper cell type 1 (Th1)-driven cytokines and thus differs profoundly from normal testis or inflammatory lesions associated with hypospermatogenesis.



T cells are known to be the major component of inflammatory infiltrates associated with either hypospermatogenesis or testicular cancer. It has previously been reported that B cells are only involved within infiltrates of seminoma samples, but this has not been investigated further.



Immunohistochemical characterisation (IHC) of infiltrating immune cells and RT-qPCR-based analysis of corresponding cytokine microenvironments was performed on different testicular pathologies. Testicular biopsies, obtained from men undergoing andrological work-up of infertility or taken during surgery for testicular cancer, were used in this study. Samples were grouped as follows: (i) normal spermatogenesis (n = 18), (ii) hypospermatogenesis associated with lymphocytic infiltrates (n = 10), (iii) samples showing neoplasia [germ cell neoplasia in situ (GCNIS, n = 26) and seminoma, n = 18].



IHC was performed using antibodies against T cells (CD3+), B cells (CD20cy+), dendritic cells (CD11c+), macrophages (CD68+) and mast cells (mast cell tryptase+). Degree and compartmental localisation of immune cells throughout all groups analysed was evaluated semi-quantitatively. RT-qPCR on RNA extracted from cryo-preserved tissue samples was performed to analyse mRNA cytokine expression, specifically levels of IL-1β, IL-6, IL-17a, tumour necrosis factor (TNF)-α (pro-inflammatory), IL-10, transforming growth factor (TGF)-β1 (anti-inflammatory), IL-2, IL-12a, IL-12b, interferon (IFN)-γ (Th1-driven), IL-4, IL-5, IL-13, IL-23a (Th2-driven), CXCL-13, CXCL-10 and CCL-5 (chemokines).



This is the first study showing a direct linkage between the distribution pattern of immune cells in hypospermatogenesis versus testicular cancer and analysis of a wide range of 17 related cyto- and chemokines. A fundamental difference between testicular inflammation patterns associated with different testicular inflammatory conditions either containing or lacking neoplastic cells was demonstrated. In hypospermatogenesis, T cells were detected, whereas B cells and dendritic cells were almost absent. Within GCNIS and seminoma, in addition to T cells, high numbers of dendritic cells and B cells were found, the latter additionally organised in cell clusters, whereas mast cells were absent. Transcripts encoding pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), anti-inflammatory cytokines (TGF-β1), Th1-driven cytokines (IL-2 and IFN-γ) as well as chemokines (CXCL-13, CXCL-10 and CCL-5) were all significantly increased in testicular germ cell neoplasia (P ≤ 0.01), suggesting the presence of a pro-tumorigenic environment. In contrast, Th2-related cytokines (IL-5, IL-13 and IL-23a) characterised the environment within samples showing normal spermatogenesis as well as hypospermatogenesis. One of the most important outcomes is the pivotal role of IL-6 in testicular cancer that opens potential novel diagnostic and/or immune-therapeutic perspective for testis cancer.



Testicular tissue composed of immune cells as well as other somatic cells and germ cells does not allow identification of specific cytokine sources or single cell types, being responsible for establishing the overall cytokine environment. In this study, laser-assisted microdissection did not reach the required efficiency for RT-qPCR analyses. Therefore, in vitro models would be suggested for addressing the above-mentioned issue. Conclusions about cytokine levels in testes with GCNIS are based on a small number of samples.



The unique B cell presence and the significantly increased expression level of IL-6 in testicular germ cell neoplasia (P < 0.001) strengthen its special role in this disease. In line with current knowledge on other types of cancer, these results underline the relevance of further investigating the potential of IL-6 as early biomarker and target for therapeutic intervention in testicular germ cell neoplasia.



This study (and B.K. in person) was supported by the Deutsche Forschungsgemeinschaft (DFG) as part of the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) on 'Molecular pathogenesis on male reproductive disorders'. T.H., H.-C.S. and M.B. were supported by the LOEWE focus group 'MIBIE' (male infertility during infection & inflammation)-an excellence initiative of the German state government of Hessen. From the Australian side, K.L. was supported by NHMRC grants (Fellowship, ID1079646 and Project, ID1081987); K.L., S.I. and M.H. received scholarship (S.I.) and research funding (K.L., M.H.) from Monash University. The project also drew support from the Victorian Government's Operational Infrastructure Support Program. The authors have no competing interests to declare.
Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia Britta.Klein@vetmed.uni-giessen.de.
Britta
B
Klein
Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany.
Thomas
T
Haggeney
Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany.
Daniela
D
Fietz
Hudson Institute of Medical Research, Wright Street, Clayton, VIC 3168, Australia.
Sivanjah
S
Indumathy
Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, VIC 3800, Australia Hudson Institute of Medical Research, Wright Street, Clayton, VIC 3168, Australia School of Clinical Sciences, Monash University, Wellington Road, Clayton, VIC 3800, Australia.
Kate L
KL
Loveland
Hudson Institute of Medical Research, Wright Street, Clayton, VIC 3168, Australia.
Mark
M
Hedger
Centre of Reproductive Medicine and Andrology, University of Muenster, Domagkstrasse 11, 48129 Muenster, Germany.
Sabine
S
Kliesch
Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
Wolfgang
W
Weidner
Institute of Veterinary Anatomy, Histology and Embryology, Justus Liebig University Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany.
Martin
M
Bergmann
Department of Urology, Pediatric Urology and Andrology, Justus Liebig University Giessen, Rudolf-Buchheim Str. 7, 35392 Giessen, Germany.
Hans-Christian
HC
Schuppe
D000328:Adult; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D016207:Cytokines / Q000378:metabolism*; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009373:Neoplasms, Germ Cell and Embryonal / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D013736:Testicular Neoplasms / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D013737:Testis / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D018417:Th1 Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D055815:Young Adult
D016207:Cytokines
RT-qPCR; TIL; cytokines; germ cell neoplasia in situ (GCNIS); immune cell; inflammation; testicular germ cell tumour; testis; tumour environment