The synthesis of the invariant natural killer (iNK) T cell agonist β-mannosylceramide along with a series of fatty amide analogues is reported.

iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo. Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, β-ManCer, a glycolipid with a β-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized β-ManCer-CD1d complexes and could inhibit β-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with β-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another β-linked glycosylceramide, β-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the β-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain β-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution.

There is growing evidence of molecular differences in prostate tumors by race or ancestry, which may contribute to the disproportionate burden of aggressive prostate cancer for African American men. Here we investigated whether RNA expression of 310 genes or non-coding RNAs that have shown differential expression (p<0.05) in prostate tumors by race/ancestry in past studies were associated with prostate tumor aggressiveness among African American men. We accessed formalin-fixed, paraffin-embedded prostate tumor tissue for 191 African American men with prostate cancer who underwent radical prostatectomy at the University of Maryland Medical Center from 1992-2021, as well as paired normal prostate tissue for a subset of the men (n=105). We generated RNA expression data using the ThermoFisher Human Clariom D array. Using Wilcoxon tests, we compared prostate tumor RNA expression by tumor aggressiveness (total Gleason score, GS, >=7 vs. <=6). The Benjamini-Hochberg False Discovery Rate approach was used to adjust for multiple comparisons. Of the 310 genes/non-coding RNAs, 110 were significantly associated with GS (p<0.05), and 65 remained significant after multiple comparison adjustment (q<0.05). Almost all (60/65) of the top findings tended to have lower expression in higher- vs. lower-grade tumors. There were several findings with q<0.01, which included B-cell CLL/lymphoma 2 (BCL2; median and IQR for GS<=6: 4.6, 4.3-4.8 and GS>=7: 4.3, 4.0-4.5; p=9.90E-05; q=0.01) and zinc finger homeobox 3 (ZFHX3; median and IQR for GS<=6: 4.0, 3.7-4.7 and GS>=7: 3.7, 3.5-4.0; p=1.30E-04; q=0.01). Notably, both genes were found to have lower expression in prostate tumor tissue in African American men compared to European American men in previous studies. Therefore, the direction of our findings with GS (lower expression for higher-grade tumors) is consistent with a potential role of these genes in prostate cancer disparities. The genes that had higher expression in higher- vs. lower-grade tumors in our study included several genes that have shown higher expression in tumor tissue from African American men than European American men in previous studies. These included splicing factor 1 (SF1; median and IQR for GS<=6: 3.5, 3.1-5.1 and GS>=7: 4.6, 3.3-7.4; p=5.60E-03; q=0.04), uroplakin 3B-like (UPK3BL; median and IQR for GS<=6: 6.1, 4.9-7.7 and GS>=7: 7.4, 5.5-9.3; p=7.20E-03; q= 0.04), and RNA binding motif protein 6 (RBM6; median and IQR for GS<=6: 9.6, 8.2-10.7 and GS>=7: 10.3, 8.9-12.1; p=0.01; q=0.05). While replication is needed, these findings add to growing evidence that RNA expression differences in prostate tumors by race or ancestry may contribute to prostate tumor aggressiveness and prostate cancer disparities. Citation Format: Jessica Yau, Ebuka Onyenobi, Yuji Zhang, Teklu B. Legesse, Gary Rose, Guangjing Zhu, Allen Burke, Ashley Cellini, Kimberly Clark, Nicholas Ambulos, Jing Yin, Søren M. Bentzen, Arif Hussain, Joanne Dorgan, Lorelei A. Mucci, Kathryn Hughes Barry. RNA expression markers with differential expression by race/ancestry are associated with prostate tumor aggressiveness among African American men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1897.

Invariant natural killer T cells (iNKT) are T cells that recognize a diverse repertoire of structurally distinct lipid antigens, instead of peptides, presented by the non-classical major histocompatibility complex molecule, CD1d. Agonists of iNKT cells that induce strong anti-tumor immunity in vivo include alpha-galactosylceramide (aGC) and beta-mannosylceramide (bMC). Previous studies have shown that altering the lipid structures of aGC resulted in different cytokine profiles and anti-tumor properties. In this study, we aimed to examine the effect of altering the lipid structure of bMC on cytokine profiles and anti-tumor activity. The ceramide structure of bMC was replaced with structures that had been reported to significantly alter the activity of aGC. We examined the analogues of bMC whose acyl chain was altered to a lyso, phenylfluoro, phenyl-O-phenylfluoro, or shorter unsaturated form (C20:2). Alpha-mannosylceramide (aMC) and lyso-alpha-mannosylceramide were also studied. An in vitro stimulation assay of two iNKT cell hybridomas, DN32.D3 and 24.8A, was used to determine whether the analogues can be recognized by TCRs of iNKT cells when presented by CD1d. aMC exhibited 8-fold less activity than bMC in the hybridoma assay. An in vivo CT26 lung metastasis model was used to measure the anti-tumor activity of the analogues. Surprisingly, the analogues, besides aMC, were not recognized by the iNKT hybridoma cell lines and appear to have little or no effect on tumor growth typically seen with aGC and bMC in the CT26 lung metastasis model. These results demonstrate significant differences between aGC and bMC in how the structural changes in the lipid portion affect the anti-tumor biological activity.