The Brugia malayi filarial antigens recognised preferentially by sera from an endemic normal population are considered to be potential vaccine candidates. By immunoscreening the cDNA library of the infective L3 stage of B. malayi with pooled endemic normal sera, a cDNA clone Bm-SL3 was identified. Analysis of sera from different patient groups with the rBm-SL3 protein showed it to be highly reactive with endemic normal sera compared to its reactivity with microfilaraemic and non-endemic normal sera. The immunoprotective efficacy of the rBm-SL3 antigen against B. malayi filarial infection was evaluated in susceptible host jirds (gerbils) (Meriones unguiculatus). Jirds immunised with the rBm-SL3 antigen showed 68% cytotoxicity against microfilariae and 67-69% cytotoxicity against infective larvae in in-vitro antibody-dependent cellular cytotoxicity assays and in-situ micropore chamber methods. Analysis of IgG subclasses against Bm-SL3 revealed a significant increase in IgG1 and IgG2 antibodies in endemic normal sera compared with other groups. Lymphocyte proliferation to Bm-SL3 was significantly higher in the endemic normal group compared with that in clinical and microfilarial carriers (p < 0.001). Significantly enhanced levels of IFN-gamma in the culture supernatant of peripheral blood mononuclear cells of endemic normal sera after stimulation with Bm-SL3 suggest that the cellular response in this group may have a Th1 bias.
Lymphatic filariasis caused mainly by infection fromW. bancrofti andB. malayi remains a major cause of clinical morbidity in tropical and subtropical countries. Analysis ofB. malayi mf, infective larval and adult worm lysates for the activity of enzymes led to the demonstration of activities of three key enzymes of carbohydrate metabolism viz., Malate dehydrogenase (MDH), Malic enzyme (ME) and Glucose-6-phosphate dehydrogenase (G6PDH) in all the three stages of the parasite. The specific activity of all the three dehydrogenases was significantly high in mf lysate compared to their activity in lysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel to their activity inlysates of the other two stages (P<0.001). Analysis by native polyacrylamide gel electrophoresis (PAGE) using 7.5% non-gradient gel showed the presence of two isoforms of each of the three enzymes (MDH, ME & G6PDH) in mf lysate, while only one form of each enzyme was present in L(3) larval and adult worm lysates. Further proteolytic enzyme activity was demonstrated both in microfilarial and infective larval lysates ofB. malayi. While both mf and L(3) larval lysates showed optimal protease activity at alkaline pH of 9.0, the mf lysate showed increased activity also at pH 3.0. The infective larval lysate was markedly inhibited by Tosylamide-L-Phenylalanine chloromethyl ketone (TPCK), a thiol protease inhibitor, while the protease activity in mf lysate was significantly inhibited by both TPCK and a serine protease inhibitor Phenyl Methyl Sulphonyl Flouride (PMSF). In sodium do-decyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using gelatin copolymerized gel, the microfilarial lysate showed 3 protease molecules of 40 kDa, 180 kDa and 200 kDa and the L(3) larval lysate had 6 protease molecules of 18, 25, 37, 49, 70 and 200 kDa size.
Responsiveness to immunotherapies is limited in melanoma to subsets of patients. Despite attempts to improve responsiveness to immunotherapies by testing combination therapies, patients often experience significant adverse events that require the termination of treatment. Although causes of nonresponsiveness remain unclear and the subject of active investigation, a commonality among refractory tumors is low pretreatment levels of tumor-infiltrating lymphocytes (TILs). Thus, studies aiming to elucidate the interactions between melanoma and immune cells and to increase TIL levels are anticipated to improve therapies. We previously reported that fucosylation (the post-translational modification of proteins with the dietary plant sugar L-fucose), is altered in melanoma. Increasing fucosylation levels in melanoma—genetically or with oral L-fucose—suppresses tumor growth, metastasis, and increases TILs in immune competent mouse models of melanoma (Lau et al., 2015). The mechanisms underlying how melanoma fucosylation appears to trigger antitumor immunity are unclear. Here, we identified TIL subpopulations triggered by fucosylation to mediate tumor suppression and a key molecular mechanism. In NRAS- and BRAF-mutant melanoma models, dietary L-fucose suppressed tumor growth by ~50-60% and increased TILs by ~10-50-fold. Of all TIL subpopulations examined, CD3+ T cells were most increased by L-fucose, doubling or increasing by 15-fold, in NRAS- or BRAF-mutant models, respectively. Depletion of CD4+ T cells abrogated L-fucose-triggered TIL increases and tumor suppression, indicating that CD4+ T cells are central for antitumor immune effects of L-fucose. We identified the Class II MHC protein HLA-DRB1 as expressed and fucosylated in melanoma cells and confirmed its requirement for L-fucose-triggered, CD4+ T cell-dependent tumor suppression in vivo. Roles of HLA-DRB1 fucosylation in melanoma, effects on CD4+ T cell biology, and how L-fucose can enhance immunotherapies will be discussed. Our studies highlight how L-fucose supplementation can render tumors “immune hot” and represents a potential therapeutic strategy for improving immunotherapies. Citation Format: Daniel K. Lester, Matt Mercurio, Pasquale Innamorato, Kodumudi Krithika, Williamson Danial, Watson Gregory, Pilon-Thomas Shari, Messina Jane, Kerri L. Thomas, Susan McCarthy, Joseph Markowitz, Robert Haltiwanger, Eric Lau. Using L-fucose to render melanomas immune hot: Roles of melanoma HLA-DRB1 and CD4+T cell-mediated antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B24.
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