4.1 The effect of Hp hsp60 on immune cells.
4.1.1 Hp hsp60 inhibits the proliferation of PBMC and jurkat cells, whereas has
no effect on CD3+ T cells.
As mentioned before, we thought that Hp hsp60 might have the immune modulation ability. To test this, we used PBMC and CD3+ T cells as our targets.
PBMC were composed of many kinds of immune cells from both innate immunity, which includes monocytes, macrophages, dendritic cells, and NK cells, and adaptive immunity, such as T and B lymphocytes. Because of this feature, we used it to mimic the infection area. T cells were important immune responses. They are responsible for many immune cell activation such as APCs and B cells. Thus, we investigate CD3+ T cells in our model. We also used Jurkat cells as model. Jurkat cells are acute T cell leukemia cells and are usually used for the investigation of T cell signal transduction.
To monitor the proliferation of these immune cells, we cultured 2*105/well (106/ml) of PBMC and CD3+ T cells in the anti-CD3 mAb-pre-coading 96 well plate with culture medium (see 3.2.9) for 4 days. Jurkat cells (2*104/well, 105/ml) were cultured in a 96-well plate for 2 days. Each well was added with 40 μl MTS and incubated at 37 ℃ for with 4 hr.
The relative OD490 showed that the proliferation of PBMC was strongly inhibited by Hp hsp60 about 30% compared with the anti-CD3 activated, Hp- hsp60 untreated group, even with the TCR (T cell reception) stimulation (Figure 5).
Different concentration of Hp hsp60 (1 μg/ml, 5 μg/ml, and 10 μg/ml) all have this inhibitory effect. In Jurkat cells, the inhibition rate only was 4% and only high dose (10 μg/ml) has effect (Figure 7). However, high dose Hp hsp60 seemed to have no effect on CD3+ T cells (Figure 6).
4.1.2 Hp hsp60 inhibit the cell proliferation neither by apoptosis nor by necrosis.
Since the cell proliferation induced by TCR stimulation was inhibited by Hp hsp60, we wanted to demonstrate the mechanism of this inhibition. The cell
proliferation inhibition can be divided into two major possibilities: cell cycle arrest and cell death. The cell death is also divided into two possibilities: apoptosis and necrosis. To find out the possibility, we use the Annexin V-FITC/ PI double staining assay. Annexin V is characterized by binding to the translocated PS (phospholipid phosphatidylserine) on the cell membrane which is a feature of early apoptosis. PI is a DNA intercalating agent and a fluorescent molecule. Combine with these two factors, we can determine the cell physiological stage including the apoptosis cells, necrosis cells, and alive cells. The analysis rule shows in Figure 8. In this experiment, we used
the same culture condition as used in MTS assay.
All the anti-CD3 activated, Hp hsp60 untreated cell group (Figure 9, lane 2) and the activated, Hp hsp60-treated groups (Figure 9, lane3, 4, and 5) exhibited no difference in the cell percentage of apoptosis, necrosis and alive cells. It means that the cell proliferation inhibition might not due to cell death, but cell cycle arrest. On the other hand, in CD3+ T cell experiment, the cell percentage of apoptosis, necrosis and alive cells in the activated, untreated cell group (Figure 10, lane2) were no difference compared with those in the activated, Hp hsp60-treated groups (Figure 10, lane3). This result was fit with the result in MTS assay.
4.2 The effect of Hp hsp60 on Treg differentiation
4.2.1 Hp hsp60 increase the percentage of Treg in CD4+ T cells
Since Hp hsp60 inhibits the cell proliferation of PBMC, we wanted to know how Hp hsp60 inhibits the proliferation. As mentioned before, Treg are a powerful immune modulation cells that suppress almost all kinds of immune cells. If Hp hsp60 inhibit the cell proliferation via Terg, the number of Treg should be increase. To clarify this possibility, we cultured 106 cells/ml PBMC and CD3+ T cells with 100 U/ml rHuman IL-2 in anti-CD3 mAb- pre-coaded 24 well plate, respectively. 10 μg/ml Hp hsp60
was used for this experiment. After treatment for 6 days, cell were harvested and stained with anti-human CD4-FITC and anti-human CD25-PE. After detected with cytometry by FL1 and FL2 channels, the cell percentage was analysized as shown in Figure 11.
In PBMC, Hp hsp60 enhanced the percentage of CD4+CD25+ T cell in CD4+ T cell under the stimulation of TCR (Figure 12). Although the percentage of
CD4+CD25+ T cell also increased in CD4+ T cell without TCR stimulation, however, this is no significant that compared with the untreated group (Figure 12). Similar result was happened in CD3+ T cells. Compared with the untreated group, the Hp hsp60 enhanced the percentage of CD4+CD25+ T cell in CD4+ T cell with the help of TCR stimulation (Figure 13B), however, the increase percentage was not significant when cultured without the present of anti-human CD3 mAb (Figure 13A). Collect these finding together, we found that Hp hsp60 has the ability to increase the number of CD4+CD25+ T cell with the TCR stimulation.
4.2.2 Hp hsp60 induces the foxp3 mRNA expression under the TCR stimulation
Although Treg express both CD4 and CD25 on the surface at the same time, however, CD4+CD25+ T cells do not equal to Treg. In this experiment, we wanted to
confirm that whether these increased CD4+CD25+ T cells were Treg. foxp3 is a key factor for differentiation and suppression function of Treg. Therefore, we used
real-time PCR to detect the mRNA expression level of the foxp3 with the same culture condition as the experiment in 4.2.1. In Figure 14 B, we found that foxp3 mRNA expression level in activated, Hp hsp60 treated CD3+ T cells could be induced up to 2.5 times compared with the untreated group. However, the phenomenon was not present without the signal of TCR (Figure 14 A).