SUMMARY Hormone suppression given before or after cytotoxic treatment stimulates recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated 6 of them with GnRH-antagonist (GnRH-ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with GFP-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared to radiation-only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the sperm of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (a) significant increases (~20%) in the volume and weight of the testes compared to the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (b) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24 weeks (9.6% vs. 2.9%; P=0.05) and 44 weeks (16.5% vs. 6.1%, P=0.055); (c) detection of lentiviral sequences in the sperm or testes of five of the GnRH-ant–treated monkeys; and (d) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

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Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2–selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

DCs play critical roles in promotion of autoimmunity or immune tolerance as potent APCs. In our anti-GBM GN model, WKY rats develop severe T cell-mediated glomerular inflammation followed by fibrosis. A DC-like cell population (CD8αα(+)CD11c(+)MHC-II(+)ED1(-)) was identified in the inflamed glomeruli. Chimera experiments demonstrated that the CD8αα(+) cells were derived from BM. The CD8αα(+) cells infiltrated glomeruli at a late stage (Days 28-35), coincident with a rapid decline in glomerular inflammation before fibrosis. The CD8αα(+) cells isolated from inflamed glomeruli were able to migrate rapidly from the bloodstream into inflamed glomeruli but not into normal glomeruli, suggesting that the migration was triggered by local inflammation. Despite high-level expression of surface and cellular MHC class II molecules, in vitro experiments showed that this CD8αα(+) DC-like cell induced apoptosis but not proliferation in antigen-specific CD4(+) T cells from T cell lines or freshly isolated from lymph nodes; they were not able to do so in the absence of antigens, suggesting induction of apoptosis was antigen-specific. Furthermore, apoptotic T cells were detected in a large number in the glomeruli at Day 32, coincident with the infiltration of the cells into glomeruli, suggesting that the cells may also induce T cell apoptosis in vivo. A potential role of this CD8αα(+) DC-like population in peripheral immune tolerance and/or termination of autoimmune inflammation was discussed.

Although gonadotropins and androgen are required for normal spermatogenesis and both testosterone and follicle-stimulating hormone (FSH) are responsible for the inhibition of spermatogonial differentiation that occurs in irradiated rats, it has been difficult to identify the specific genes involved. To study specific hormonally regulated changes in somatic cell gene expression in the testis that may be involved in these processes, without the complication of changing populations of germ cells, we used irradiated LBNF(1) rats, the testes of which contain almost exclusively somatic cells except for a few type A spermatogonia. Three different groups of these rats were treated with various combinations of gonadotropin-releasing hormone antagonist, an androgen receptor antagonist (flutamide), testosterone, and FSH, and we compared the gene expression levels 2 wk later to those of irradiated-only rats by microarray analysis. By dividing the gene expression patterns into three major patterns and 11 subpatterns, we successfully distinguished, in a single study, the genes that were specifically regulated by testosterone, by luteinizing hormone (LH), and by FSH from the large number of genes that were not hormonally regulated in the testis. We found that hormones produced more dramatic upregulation than downregulation of gene expression: Testosterone had the strongest upregulatory effect, LH had a modest but appreciable upregulatory effect, and FSH had a minor upregulatory effect. We also separately identified the somatic cell genes that were chronically upregulated by irradiation. Thus, the present study identified gene expression changes that may be responsible for hormonal action on somatic cells to support normal spermatogenesis and the hormone-mediated block in spermatogonial development after irradiation.