Oral composition comprising lactic acid bacteria for regulating immune responses and methods related thereto
A composition and technology of Lactobacillus, applied in the direction of blood/immune system cells, medical raw materials derived from mammals, drug combinations, etc., can solve the problems of ignorance of the potential mechanism of NK cell immune regulation
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Embodiment 1
[0233] Example 1: Materials and methods used in Examples 2-3
[0234] Cell Lines, Reagents and Antibodies
[0235] Human immune cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) (Gemini Bio-Products, CA). Oral squamous cancer stem cells (OSCSC) were isolated from tongue tumors of oral cancer patients at UCLA School of Medicine, and were supplemented with 10% FBS (Gemini Bio-Products, CA), 1.4% antibiotic antimycotic (antimycotic), 1% acetone sodium bicarbonate, 1.4% MEM non-essential amino acids, 1% L-glutamine, 0.2% gentimicin (GeminiBio-products, CA) and 0.15% sodium bicarbonate (Fisher Scientific, PA) in RPMI 1640 to cultivate.
[0236] Antibody against CD16 was purchased from Biolegend (San Diego, CA, USA). Recombinant IL-2 was obtained from NIH-BRB. Antibodies against isotype control, MHC-I, CD45 (human), CD45 (mouse), CD3, CD16, CD56, CD8, HLADR and CD11b were purchased from Biolegend (San Diego, CA). Human NK purification kit was obta...
Embodiment 2
[0279] Example 2: Probiotic bacteria activate NK cells
[0280] Probiotic bacteria induce cytokine secretion but have no effect on cytotoxicity
[0281] To explore the effect of probiotics on NK cell function, NK cells were cultured with or without sAJ2 under different activation conditions. After treatment with sAJ2 probiotics, NK cells retained as controls or activated with IL-2 or IL-2+anti-CD16 mAb induced higher levels of IFN-γ and IL-10 ( figure 1 ). Although activated NK cells secreted IFN-γ, treatment of activated NK cells with sAJ2 bacteria further significantly increased the level of IFN-γ ( figure 1 A). Treatment with sAJ2 induced IL-10 secretion under all NK conditions, especially for control NK cells (not activated) ( figure 1 B).
[0282] Although sAJ2 bacteria induced cytokine secretion in NK cells, they had no effect on the cytotoxic function of NK cells ( figure 2 ). Untreated NK cells as well as activated NK cells did not exhibit significant changes...
Embodiment 3
[0289] Example 3: Osteoclasts and monocytes combined with probiotic bacteria to expand NK cells
[0290] sAJ2 in combination with osteoclasts expands NK cells but not T cells, maintaining them for longer periods
[0291] NK cells were isolated from healthy donor PBMCs and NK purity was determined using CD3 and CD16 / CD56 antibodies before being cultured. NK cells were then activated with anti-CD16 mAb and IL-2 18-20 hours before co-culture of NK cells with sAJ2 alone ("control") or sAJ2 in combination with osteoclasts ("+osteoclasts") ( Figure 8 ). NK cells treated with sAJ2 failed to expand and maintain NK cell purity over time, whereas NK cells cultured with sAJ2 and osteoclasts maintained NK purity and expansion for prolonged periods. The combination of OC and sAJ2 preferentially expanded NK cells while maintaining a low proportion of T cells throughout the culture ( Figure 8 ).
[0292] Expanded NK cells are highly functional with respect to both cytotoxic and cytok...
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