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Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.

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This study compared the YeastOne and reference CLSI M38-A2 broth microdilution methods for antifungal susceptibility testing of Aspergillus species. The minimum inhibitory concentrations (MICs) of antifungal agents against 100 isolates, comprising 31 Aspergillus fumigatus (5 TR34/L98H isolates), 23 Aspergillus flavus, 13 Aspergillus terreus, and 10 Aspergillus niger clinical isolates, and 23 A. fumigatus environmental isolates (19 TR34/L98H isolates), were determined. The overall agreement (within two twofold dilutions) between the two methods was 100%, 95%, 92%, and 90% for voriconazole, posaconazole, itraconazole, and amphotericin B, respectively. The voriconazole geometric mean (GM) MICs from both methods were nearly identical for all isolates, whereas the itraconazole and posaconazole YeastOne GM MICs were about one dilution lower than the reference GM MICs. In contrast, the amphotericin B YeastOne GM MIC was 3.3-fold higher than the reference GM MIC. Of 24 TR34/L98H A. fumigatus isolates, the categorial agreement (classified according to the EUCAST clinical breakpoints) was 100%, 91.7%, and 83.3% for itraconazole, voriconazole, and posaconazole, respectively. Of four A. niger isolates, itraconazole MICs were read as > 8 mg/L by M38-A2 due to trailing growth in wells containing itraconazole concentrations of 0.5 to 8 mg/L, whereas the corresponding itraconazole MICs by YeastOne were all u2264 0.25 mg/L, without trailing growth. These data suggested that YeastOne could be a suitable alternative for azole susceptibility testing of Aspergillus species and for detecting TR34/L98H A. fumigatus. Nevertheless, YeastOne tended to yield higher amphotericin B MICs and failed to detect A. niger isolates which exhibited trailing growth with itraconazole.

Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, we show that the miR-23~27~24 clusters regulate multiple aspects of T cell biology, particularly helper T (Th) 2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23~27~24 clusters revealed that miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, although overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17, and induced regulatory T cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest that tight regulation of miR-23~27~24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.