【小编误打误撞进入此行业之后，经常听很多研发人员讲起用我们的PBMC做MLR, SEB, ADCC之类的实验，但具体对于这类实验的目的是什么，PBMC是如何用于此类实验之类的问题往往是一知半解，为了给大家提供更好的服务，这段时间我们团队也开始在学习这方面的内容，不过可能也是毕业有些年头了，学习能力退化的比较厉害，因而也逼着自己写写公众号，将看到的比较不错的文献，微信文章整理整理，分门别类一下，希望有一点点抛砖引玉的作用吧】
Mixed lymphocyte reaction (MLR).
Dendritic cells (DCs) were generated byculturing monocytes isolated from PBMCs using a monocyte purification kit invitro for 7 days with 500 U/mL IL- 4 and 250 U/mL GM-CSF.
CD4+ T cells (1 x105) and allogeneic DCs (1 x 104) were co-cultured with or without dosetitrations of nivolumab added at the initiation of the assay.
After 5 days, IFN secretion in culture supernatants was analyzed by ELISA, and cells were labeled with 3H-thymidine for an additional 18 hours to measure T-cell proliferation.
Staphylococcal enterotoxin B(SEB) stimulation of PBMC.
PBMCs from healthy human donors (N = 18) were cultured for 3 days with nivolumab or an isotype control antibody (20 μg/mL) at the initiation of the assay together with serial dilutions of SEB. Interleukin-2(IL-2) levels in culture supernatants were measured by ELISA analysis.
Antigen-specific recall response in vitro.
In a cytomegalovirus (CMV)-restimulationassay, 2 x 105 PBMCs from a CMV-positive donor were stimulated using lysate of CMV-infected cells, with serial dilutions of nivolumab added at the initiationof the assay. After 4 days, supernatants were assayed for IFNγ.
Suppression assay with regulatory T cells (Treg).
CD4+CD25+ Tregs and CD4+CD25- responder Tcells were purified from PBMC (CD4+CD25+ Treg isolation kit). In an allogeneicMLR assay, Tregs (5 x 104) were co-cultured with 1 x 105 responder T cells and2 x 104 monocyte-derived DCs, with 20 μg/mL nivolumab.
After 5 days, IFNγ production was assessed in supernatants,and cells were labeled with 3H-thymidine for an additional 18 hours for proliferation analysis.
Antibody-dependent cell-mediated cytotoxicity (ADCC).
PBMCs were incubated overnight with 50 U/mL IL-2 and used as effector cells.
Activated CD4+ T cells labeled with BATDA reagent were used as target cells at an effector-to-target cell ratio of 50:1. Serial dilutions of nivolumab or positive control (anti-MHC I antibody) were added; the cells were incubated for 3 hours at 37ºC.
To measure cytotoxicity,supernatant was mixed with Europium-solution and read using a RUBYstar Model460 microplate reader (BMG LABTECH).
In an allogeneic MLR, PD-1 blockade with nivolumab systematically resulted in a titratable enhancement of IFN release, and in some donor T cell/DC pairs, enhanced T-cell proliferation was observed(Fig. 2A).
Nivolumab also enhanced IL-2 secretion over isotype control inresponse to SEB using PBMCs (Fig. 2B).
Using a CMV- restimulation assay, nivolumab, compared with an isotype control, resulted in a concentration-dependent augmentation of IFNγ secretion from CMV-responsive donors (Fig. 2C).
While PD-1 expression can be observed in T cells prior to stimulation by allogeneic DCs, SEB or antigen,PD-1 expression is up regulated after T-cell activation in all of these assays (Figure S4A, 4B and data notshown).
In addition, PD-L1 expression and up regulation can be observed in multiple cell subsets in these assays (Figure S4B and data not shown).
As Tregs also express PD-1, nivolumab was assessed in an allogeneic MLR in which Tregs suppressed the proliferation and cytokine secretion of purified CD4+CD25- responder T cells, which were stimulated by allogeneic DCs.
In this assay, nivolumab completely restored proliferation and partially restored IFN release by the alloreactive T cells(Fig. 2D).
Taken together, these data show that nivolumab can, at very low concentrations (~1.5 ng/mL), enhance T-cellreactivity in the presence of a T-cell receptor stimulus. However, nivolumab had no effect in the absence of antigen or T-cell receptor stimulus. Specifically, there was no significant release of inflammatory cytokines, including IFNγ, TNF-α, IL-2, IL-4, IL-6, or IL-10, from unstimulated whole blood after co-incubation with nivolumab. In contrast, positive-control anti-CD3 antibody potently increased cytokine release (Table S2). These results demonstrate that nivolumab does not cause non- specific lymphocyte activation.
Lastly, the ability of nivolumab (tested from 0.003 μg/mL to 50 μg/mL) to mediate ADCC activity in vitro was tested. Using IL-2-activated PBMCs as a source of natural killer (NK) cells and activated human CD4+ T cells expressing high levels of cell-surface PD-1 as target cells, nivolumab (IgG4 [S228P]) did not mediate ADCC (Fig. 3). Limited ADCC activity was observed with the parental form of nivolumab, an IgG1 antibody purified from hybridoma supernatant at high antibody concentrations, whereas positive control anti-MHC-class I antibody was able to mediate ADCC of T cells at lowerantibody concentrations. In addition, nivolumab did not mediate complement-mediated cytotoxicity (CDC) of activated human CD4+ T cells in thepresence of human complement (data not shown).
MLR: 我司有已经验证过，配对好的PBMC个体，一组提取CD14分化DC，一组提取对应T cell，并可以持续稳定提供固定个体；
CMV positive donor PBMC；
Treg：可提供同一个体 Treg和剩余的T cell，可根据需要进行定制化分装；
Isotype Control Antibody：Anti-HEL (Hen Egg Lysozyme) without anyknown cross-reactive to human.
1.血液免疫学细胞——PBMC及亚型（CD3,CD4,CD8,CD56,CD14等），serum，plasma；Cord Blood；Bone Marrow；
2. 阳性对照抗体——Anti-PD1, Anti-PDL1，Anti-CD47，Anti-Tigit，Anti-Lag3，Anti-Tim3等；