Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

Metabolic transitions define spermatogonial stem cell maturation.

STUDY QUESTION: Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? SUMMARY ANSWER: Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. WHAT IS KNOWN ALREADY: Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. STUDY DESIGN, SIZE, DURATION: To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. LARGE SCALE DATA: New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. LIMITATIONS, REASONS FOR CAUTION: Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.

Human papillomavirus detection in moroccan patients with nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor which arises in surface epithelium of the posterior wall of the nasopharynx. There's is evidence that Epstein Barr virus (EBV) is associated to NPC development. However, many epidemiologic studies point to a connection between viral infections by the human papillomavirus (HPV) and NPC. METHOD: Seventy Moroccan patients with NPC were screened for EBV and HPV. EBV detection was performed by PCR amplification of BZLF1 gene, encoding the ZEBRA (Z Epstein-Barr Virus Replication Activator) protein, and HPV infection was screened by PCR amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 35, 45 and 59. RESULTS: The age distribution of our patients revealed a bimodal pattern. Sixty two cases (88.9%) were classified as type 3 (undifferentiated carcinoma), 6 (8.6%) as type 2 (non keratinizing NPC) and only 2 (2.9%) cases were classified as type 1 (keratinizing NPC). EBV was detected in all NPC tumors, whereas HPV DNA was revealed in 34% of cases (24/70). Molecular analysis showed that 20.8% (5/24) were infected with HPV31, and the remaining were infected with other oncogenic types (i.e., HPV59, 16, 18, 33, 35 and 45). In addition, statistical analysis showed that there's no association between sex or age and HPV infection (P > 0.1). CONCLUSION: Our data indicated that EBV is commonly associated with NPC in Moroccan patients and show for the first time that NPC tumours from Moroccan patients harbour high risk HPV genotypes.

IL-7 enhances survival of human CD56bright NK cells.

Lymphoid differentiation and activation critically depend on cytokine stimulation and the interleukin-7 (IL-7) signaling in particular. Although it has been demonstrated that IL-7 may play a role in natural killer (NK) cell maturation, the effect of IL-7 stimulation on mature human NK cells has not been studied. We, therefore, investigated the expression and functional activity of IL-7Ralpha on mature NK populations from adult blood. In this article, we demonstrate that IL-7Ralpha is specifically expressed in the CD56bright noncytotoxic cytokine-producing NK subset. Importantly, this expression is thymus independent, contrary to what is observed in mice. In addition, we show that IL-7Ralpha is expressed at higher levels on NKG2A+CD56bright NK cells. In contrast to IL-15 stimulation, IL-7 does not increase NK cell cytotoxicity, interferon-gamma production, or the expression of activation markers, indicating that these cytokines play different functions in NK homeostasis and activation. However, IL-7 promotes the survival of the CD56bright NK subset and inhibits apoptosis by increasing BCL2 expression. These data should be taken into account when considering the clinical use of IL-7, particularly after stem cell transplantation.

Binding of thymoglobulin to natural killer cells leads to cell activation and interferon-gamma production.

Thymoglobulin is an antithymocyte globulin preparation used in hematopoietic stem cell transplantation (HSCT) to prevent rejection and graft-versus-host disease. Because natural killer (NK)-cell alloreactivity improves HSCT outcome, but only in patients receiving thymoglobulin, we investigated the in vitro effects of thymoglobulin on purified NK cells. Thymoglobulin binding to NK cells and NK-cell activation were assessed by flow cytometry. NK surface targets for thymoglobulin were determined by competition inhibition assays using monoclonal antibodies. Chromium 51 (Cr) release assay, Annexin V combined with 7-amino-actinomycin D staining, and carboxyfluorescein diacetate succinimidyl ester staining were used to study cytotoxic activity, apoptosis/cell death, and NK-cell proliferation, respectively. Interferon (IFN)-gamma production was determined by ELISA. Thymoglobulin, thymoglobulin derived-F(ab')2 fragments as well as rabbit IgG bound NK cells, and competed strongly with anti-CD16. Thymoglobulin enhanced the expression of activation (CD69 and NKG2D) and degranulation (CD107a) markers on NK cells. It competed with CD18 binding and decreased NK activity, but not interleukin-15-induced killer activity. Effects on apoptosis/cell death and proliferation were minimal. F(ab')2 fragments and rabbit IgG strongly induced IFN-gamma production by NK cells. Thymoglobulin binds to NK cells by CD16 by its variable and constant regions. The decrease in NK-cell cytotoxic activity is restored by interleukin-15, and contrasts sharply with the induction of activation, degranulation, and IFN-gamma production. These data support the hypothesis that thymoglobulin treatment is required to observe the improvement in HSCT outcome by NK-cell alloreactivity.

[Neonatal immunology and cord blood transplantation]. / Immunologie néonatale et greffe de sang de cordon.

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "naïve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although naïve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.

Antibody responses to recombinant Epstein-Barr virus antigens in nasopharyngeal carcinoma patients: complementary test of ZEBRA protein and early antigens p54 and p138.

Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54+138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18+23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54+138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54+138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54+138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC.

Study of human leukocyte antigen class I phenotypes in Moroccan patients with nasopharyngeal carcinoma.

Previous reports demonstrated an association between the human leukocyte antigen (HLA) and risk for nasopharyngeal carcinoma (NPC) among the Chinese in Singapore, a population with a high incidence of this malignancy. In our study, we assess the association between HLA and NPC in Morocco, a median-prevalence area for this cancer, where NPC presents the particularity of also affecting young individuals. Using the standard microlympho-cytotoxicity test, we typed a total of 154 Moroccan NPC patients and 257 unrelated healthy controls for their HLA-A and B antigens. The results of these analyses show that the frequencies of HLA-A10, HLA-B13 and HLA-B18 were found to be higher in the NPC group than in the control group, whereas HLA-A9 was associated with a decreased risk. After correction for the number of specificities tested, these differences were statistically significant only for HLA-B18 (corrected p value [pc] < 0.023, relative risk [RR] = 4.14) and HLA-A9 (pc < 0.023, RR = 0.45). The comparison of the distribution of the HLA antigens in younger and older cohorts of patients shows that the incidence of HLA-A10 and HLA- B18 was higher in the older group, whereas the frequencies of HLA-A19 and HLA-B13 were significantly higher in younger patients compared with controls. The presence of both HLA-A19 and HLA-B13 phenotypes correlated with an increased risk of developing NPC among overall patients compared with controls. According to the sex distribution, increased frequency of HLA-B18 was found in male and female NPC patients compared with controls, whereas the frequency of HLA-A10 was higher only in male NPC patients compared with controls.

Antibodies to the Epstein-Barr virus transactivator protein (ZEBRA) as a valuable biomarker in young patients with nasopharyngeal carcinoma.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) generally occurs in adults, especially in high-prevalence populations such as the Chinese and Eskimos. In Maghrebian populations, young patients affected with this malignancy represent 25% of the total NPC cases. In adults with NPC, relatively high titers of IgA antibodies to the EBV viral capsid antigen (VCA) and early antigen (EA) represent important markers. However, nearly 50% of young NPC patients are negative for IgA-anti-VCA and -EA or exhibit very low titers of these antibodies. We report here that 92% of sera from young NPC patients negative for IgA-EA and 89% of those negative for IgA-VCA were positive for IgG antibodies to the EBV transactivator protein (ZEBRA) at very high titers. Our results show that in young patients with NPC these antibodies represent the most reliable marker for diagnosis and prognosis, particularly when compared with conventional NPC markers, i.e., IgA-VCA (58%) and anti-EA (25%). The titers of IgG-ZEBRA antibodies increased along with lymph node involvement only in the young patient group, suggesting a prognostic value of this marker in this patient group.