Optimization Of Culture Cytokine-induced Killer Cells And Dendritic Cells In Vitro

BackgroundHepatitis B virus is one of the most prevalent health problems in china. until now, the rate of HBsAg positive is about 7.18%, There are approximately 110 million HBV carriers in China.20 million patients need anti-viral treatment, of which 25% to 40% may develop liver cirrhosis(LC) and hepatocellular carcinoma(HCC). About 70 million patients die from hepatitis B-related complications, including LC, HCC a year. There is no methods to eradicate hepatitis B virus(HBV) from patients. The goal of therapy for hepatitis B is to improve quality of life and survival by preventing progression of the disease to cirrhosis, decompensated cirrhosis, end-stage liver disease, HCC and death. The recommended anti-viral agents to CHB include interferon and nucleoside (nucleotide) analogues(NUCs), but these drugs have limited efficacy to treat CHB patients. For example, the advantage of oral administration of nucleoside analogues is easy to use, the disadvantages of these drugs include:unclear regimen, only inhibition of HBV DNA replication, no clear role to HBV cccDNA. Although these drugs can inhibit HBV replication, if patients don't get HBeAg and/or HBsAg seroconversion, stopping them will cause a high relapse rate. Meanwhile, clinicians and patients are also puzzled by some questions, including the incidence of drug resistance and withdrawal time of these drugs. Although interferon is less likely to relapse and drug resistance, but only about 30% of patients can get benefit from it. The disadvantages of it include the application of inconvenient injection, adverse reactions, and the limitations for the patients. Therefore, there are an urgent needs to explore new methods to solve these questions.Recent studies show that the reason of persistence the chronic hepatitis B is linked to the patients' immune levels, including the reducing the number and functional defects of various immune cells, so it can not effectively clear the HBV virus. HBV can invade liver cells and replicate in liver cells, it can not be removed from the liver.It will result in inflammation of the liver, eventually LC and HCC.In order to remove viruses and reduce inflammation of the liver, in addition to the application of anti-viral treatment, We should explore another methods to increase the number of immune cells and enhance immune system function. The immune reconstitution treatment of CHB may be a new direction.CIK cell therapy is a kind of adoptive immune therapy. These cells can secrete some cytokines(eg.IFN-y) and have the ability to kill the infected cells.Currently the Ministry of Health of our country issued the third medical technology catalog of clinical application. Autoimmune cell therapy technology has been explicitly included in the third class medical technology directory. At present, some clinicians have used CIK cells to treat CHB patients.Dendritic cells (DC) is the most powerful antigen presenting cells (APC). Some studies have showed that DC can initiate immune response and effectively activate cytotoxic T lymphocyte (CTL) in Vivo and in vitro. Dysfunction of DC in CHB patients may be one of reasons of persistence of CHB.In this study, we compared different culture conditions on the effects to CIK cells, and explored optimal conditions for DC cells and the role of DC on the CIK. The aim of this study is to provide quality and reliable DC and CIK cells for future clinical applications.ObjectivesWe explored the different culture conditions on the impact on the CIK cells and DC in vitro. The aim is to find the optimal conditions of CIK cells and DC culture in order to provide quality and reliable CIK cells for clinical needs. Preliminary study on investigation about the effects of DC to CIK cells.MethodsVein blood was collected from healthy volunteers. Isolated peripheral blood mononuclear cells (PBMC) by density gradient centrifugation, CIK cells were induced by conventional methods. We used the same factors but different culture mediums (AIM V, OpTmizer) to culture CIK cells in vito. We used the same culture medium but different isotypes of anti-CD3 monoclonal antibody(UCHT1,OKT3) to induce the CIK cells. Inducing DC from monocytes needs granulocyte colony stimulating factor (GM-CSF) and interleukin 4 (IL-4). We compared different culture mediums (RPMI1640, AIM V, PAA), application of different maturation-promoting factors (cocktail, TNF-α) and different concentrations of autologous or allogeneic plasma on the impact to DC culture in vitro. HBcAg loaded DC were incubated with CIK cells.Results1. We used AIM V or OpTmizer culture medium to culture CIK cells in vitro. We found that the morphology of CIK cells vary in sizes and shapes:round, elongated, irregular shape. Part of the growth of cells form colonies. On day 14, compared OpTmizer with AIM V, the increasing times of CIK cells was 27.52:9.45(P<0.05). Analysis of cell phenotypes was different either:On day 0, the percentage of CD56+ cells is (27.17±9.32)%, on day 14, it increased to (65.20±21.67)% in AIM V, while it increased (66.97±21.42)% in OpTmizer, compared with the baseline t(P<0.05). On day 0, the percentage of CD3+CD56+ cells is (6.00±5.66)%, on day 14, it increased to (33.34±21.89)% in AIM V, while it increased (26.81±21.90)% in OpTmizer, which showed no significant difference between two culture mediums. On day 0, the percentage of CD3+CD56+ cells is (6.00±5.66)%, on day 14, it increased to (33.34±21.89)% in AIM V, while it increased (26.81±21.90)% in OpTmizer, which showed no significant difference between two culture mediums. Three cases were selected to evaluate the killing abilities to K562, when effector cells to target cells ratio (E/T) were 40:1,20:1,10:1,5:1, there is no significant difference between two culture mediums.2. We used two different anti-CD3 mAb to culture CIK cells in vitro. We found that CIK cells became larger compared to PBMC under the two culture conditions by the microscope. Cells viability is more than 90% by trypan blue assay dying, there is no significant difference between the two groups. On day 14, On day 14, compared OKT3 with UCHT1, the increasing times of CIK cells was 470.94:33.73 (P<0.05).Cell phenotypes were analyzed by flow cytometry:On day 0, the percentage of CD3+CD8+ cells is(28.82±13.36)%, on day 14, it increased to (74.94±11.45)% in OKT3, while it increased (47.27±29.38)% in UCHT1(P<0.05). On day 0, the percentage of CD56+ cells is(27.17±9.32)%, on day 14, it decreased to (16.24±13.96)% in OKT3, while it increased (38.91±25.23)% in UCHT1(P<0.05). Three cases were selected to evaluate the killing abilities to K562, when effector cells to target cells ratio (E/T) was 20:1, the killing rate were (5.01±8.69)% in OKT3 and (44.65±20.14)% in UCHT1 respectively, (P=0.078).3. We used three culture media (PAA, RPMI1640 and AIM V) to induce DC from monocytes in vitro. The morphology of immature DC (iDC) induced by three media was different:DC cultured by RPMI 1640 were shown semi-adherent state, cells were round and translucent. There were some small protrusions on the cells surface. Most cells were single, a few of cells were clusters. DC cultured by PAA were adherent and elongation. DC cultured by AIM V were shown different form, most of them are adherent cells, which wre spindle-shaped, cytoplasm extending outward. When DC became mature, the cells surface markers were analyzed by flow cytometry:DC cells purity in three different culture mediums was more than 80%. After the induction of maturation, mean fluorescence intensity (MFI) of HLA-DR, CD80, CD83, CD86 were 294.93±104.06,229.47±50.98,273.15±169.05, 93.66±26.67 in 68420.5,32073.5,12612.5,14234.5 in RPMI1640; 298.31±116.60, 132.18±15.02,175.57±91.48,142.36±17.57 in PAA and 289.55±72.65, 169.65±42.25,180.65±45.60,302.86±52.74 in AIM V.4. We used two different programs (cocktail, TNF-a) to induce the maturation of DC different programs. The cells surface markers of mDC cells were analyzed by flow cytometry:MFI of HLA-DR, CD80, CD83, CD86:294.93±104.06,229.47±50.98,273.15±169.05,93.66±26.67 in cocktail and 290.18±57.38, 130.07±25.49,91.33±4.86,83.97±28.77 in TNF-a.5. We evaluate different plasma (2% autologous plasma,10% autologous plasma 2% FBS and 10%FBS) impact on DC culture. After the induction of DC maturation, the cells surface markers of mDC cells were analyzed by flow cytometry:MFI of HLA-DR, CD80, CD83, CD86 were 407.60±130.12,139.67±84.13,177.71±37.59, 199.60±86.91 in 2% autologous plasma; 377.10±60.00,134.18±81.25,127.25±20.61, 179.66±64.85 in 10% autologous plasma; 262.15±84.16,207.28±41.56, 196.21±91.58,132.75±42.80 in 2%FBS; 294.93±104.06,229.47±50.98, 273.15±169.05,93.66±26.67 in 10%FBS. 6. Co-culture DC and CIKcells. On day 6 of DC culture, adding HBcAg into the DC, then incubated for 24 hours. Using cocktail to induce DC maturation. DC were incubated with Autologous CIK cells in a ratio of 1:10. Three days after incubation, CIK cells without DC increased 3.25±1.75-fold times,DC-CIK cells increased 3.99±1.75 times, there was significant difference (P=0.007). on day 14 of co-culture of DC-CIK and day 24 of CIK cells culture. The cells surface marker were anylazed by flow cytometry, including CD3+, CD56+, CD8+, CD3-CD56+, CD3+ CD56+ cells, there were significant differences. Detect cytotoxic T cells for HBcAg18～27V by Pentamer, PBMC, DC-CIK, CIK were:0.16%,1.98%,2.21%; HBcAg18 27Ⅰ-specific CTL, PBMC, DC-CIK, CIK were:0.17%,1.67%,1.95%.Conclusions1. AIM V and OpTmizer can proliferate CIK cells in only 1% autologous plasma into the culture, but CIK proliferation significantly decreased or even failure without plasma in culture medium. On the day 14 of culture, The cells surface marker were anylazed by flow cytometry between two mediums:the percentage of CD3+ CD56 + cells were similar. But the absolute number of CD3+ CD56+ cells, OpTmizer was significantly higher than AIM V, which shows OpTmizer can induce more numbers of CD3+ CD56+ cells than AIM V. Under both culture conditions, there was no different in killing effects to the K562. this study suggest that OpTmizer is more suitable for CIK cells proliferation in vitro.2. We used two different isotypes of anti-CD3 monoclonal antibody (UCHT1, OKT3) to induce CIK cells. The size of CIK cells was larger than PBMC by flow cytometry, which suggested that two anti-CD3 monoclonal antibody can activate cell proliferation. The proliferation numbers of CIK cells was significantly higher in OKT3 than that in UCHT1, but the main proliferation cells was CD3+ CD8+ cells, and low percentage of CD56+ cells relatively. By cytotoxicity assay, we found that on the killing effects to K562 by CIK cells induced by UCHTl was higher than that by OKT3. This study suggested that the CIK cells in a high percentage of CD56+ cells may be more important for tumor cell killing than CD3+ CD8+.3. Compared the different influence to culture DC among three culture mediums (AIM V, PAA and RPMI1640). After DC were induced to maturation, HLA-DR, CD80, CD83, CD86 of cell surface expression markers were significantly increased. But iDC cultured by AIM V expressed lower CD80 and CD83 than that of by PAA or by RPMI1640. mDC cultured by AIM V expressed the same higher CD80 and CD83 as that of by PAA or by RPMI1640. these suggested that DC cultured by AIM V can keep these cells in a good immature and mature state, so AIM V may be more suitable in culture DC than PAA or RPMI1640 in vitro.4. We compared two different programs (cocktail and TNF-a) on impact to DC maturation. Cells surface marker CD83, CD80, CD86 and HLA-DR were upregulated in mDC compared with iDC. Although the data showed that TNF-a can activate DC in some extent. But cells surface markers expression was higher in cocktail than that in TNF-a. In particular, the specificity markers CD83 expression of mature DC, cocktail group was higher than TNF-a group. The average fluorescence intensity of HLA-DR,CD80,CD83,CD86, cocktail group were also higher than that of TNF-a group. Co-express of these surface marker were higher in cocktail group than that in TNF-a group. This study suggested that Cocktail is more effective in inducing DC maturation than TNF-a.5. We compared the plasma effects on DC maturation, including 2% autologous plasma,10% autologous plasma,2% FBS and 10% FBS in this study. The results showed that there was no noticeable difference in four different serum/plasma culture DC maturation program. Cultured DC cells grew well in three groups. These results suggest that 2% autologous plasma,10% autologous plasma,2% FBS and 10% FBS can be used DC cultured cells, and the DC can be induced activation of Cocktail.6. Co-culture DC with CIK, preliminary results showed that DC can stimulate the proliferation of CIK cells, but there was no significant effects on the proliferation of cell subsets. HBcAg18～27V and HBcAg18～271-specific CTL were detected by Pentamer dying in two cases of asymptomatic HBsAg carriers, we found that the numbers of CTL were significantly increased in CIK and DC-CIK than that in PBMC.