Non-stimulated BMDC

Differences in the BMDC differentiation between the C57BL/6 wt and TNFR2^-/- background were examined using kinetics or the time points indicated. Either expression of different markers or the concentrations of soluble TNF and soluble TNFR2 in the medium were measured. All BMDC samples were non-stimulated.

3.3.1.1 Cell numbers in BMDC cultures

First, the yields of cells in the BMDC differentiation cultures of wt and TNFR2^-/- mice were analyzed. As illustrated in Figure 23, pure TNFR2^-/- BMDC cultures provided significantly reduced yields of cells on day 8 and 10.

A B

Figure 23: BMDC yields from BMDC cultures - kinetics

BMDC numbers in the petri dishes were counted on different time points during the BMDC differentiation culture period. Shown are the single values and the mean (horizontal line) of four different BMDC cultures per group representing individual mice.

3.3.1.2 Frequency of cells expressing activation markers (MHCII

CD80

CD86

) in BMDC cultures

Next, the question was addressed whether the expression of activation markers is impaired in BMDC from TNFR2^-/- mice compared to wildtype mice. TNFR2^-/- BMDC contained higher relative proportions of cells expressing the activation markers MHCII⁺, CD80⁺, and CD86⁺. This finding is visualized in Figure 24. The data are statistically significant on day 8 and 10.

BMDC number kinetics

Day 4 Day 6 Day 8 Day 10 0.0

0.5 1.0 1.5 2.0 2.5

wt TNFR2

-/-p=0.0314 p=0.0060

Yield [107 / petri dish]

Figure 24: Activation markers – BMDC cultures kinetics

The percentage of BMDC expressing the activation markers MHCII⁺, CD80⁺, and CD86⁺ were analyzed employing flow cytometry at different time points during the differentiation into dendritic cells. Shown are the single values and the mean (horizontal line) of four different BMDC cultures per group and time point representing individual mice (A). (B) illustrates the histogram of one representative culture out of four on day 10.

3.3.1.3 Frequency of MDSC in BMDC cultures

TNFR2^-/- BMDC cultures contained lower proportions of cells expressing the MDSC markers CD11b⁺, Ly6C⁺, and Ly6G^- throughout the BMDC differentiation culture period. This finding is shown in Figure 19. The data are statistically significant.

A

BMDC activation kinetics

Day 4 Day 6 Day 8 Day 10 0

10 20 30

wt TNFR2

-/-p=0.0805 p<0.0001

CD80+ CD86+ MHCII+ [% live cells]

B

3.3.1.4 Proliferation in BMDC cultures

Having shown that BMDC differentiation cultures from TNFR2^-/- mice yield reduced cell numbers compared to wildtype mice, it was next analyzed whether this is due to decreased proliferation in TNFR2^-/- BMDC differentiation cultures. TNFR2^-/- BMDC cultures on day 10 of the differentiation culture period contained slightly reduced percentages of cells that incorporated BrdU into the DNA indicating less proliferation. This finding is shown in Figure 25.

Figure 25: Proliferation in BMDC cultures

The percentages of BMDC that were positive for BrdU were analyzed employing flow cytometry on day 10. Cells were incubated with BrdU for the 24 previous hours. Shown are the single values and the mean (horizontal line) of three different BMDC cultures per group representing individual mice.

3.3.1.5 Cell death in BMDC cultures

The role of cell death for the decreased cellular yield of TNFR2^-/- BMDC was investigated next.

TNFR2^-/- BMDC did not contain altered numbers of apoptotic and necrotic cells. Data are based on flow cytometry analysis of Annexin V⁺ and 7-AAD⁺ cells. This finding is shown in Figure 26.

Figure 26: Cell death in BMDC cultures

The percentages of BMDC positive for Annexin V and 7-AAD were analyzed employing flow cytometry on day 10. Shown are the single values and the mean (horizontal line) of three different BMDC cultures per group representing individual mice.

BMDC proliferation d10

wt

TNFR2-/-0 5 10 15 20

BrdU+ [% live cells]

BMDC cell death d10

wt

TNFR2-/-0 1 2 3 4 5

Annexin V+ 7-AAD+ [% cells]

3.3.1.6 TNF concentrations in BMDC cultures

Subsequently, the concentrations of soluble and biologically active TNF in BMDC differentiation cultures of wildtype and TNFR2^-/-mice were analyzed, in order to get information about the relevance of this cytokine during the BMDC differentiation. TNFR2^-/- BMDC cultures produced significantly higher concentrations of soluble TNF on every day of the kinetic. Figure 27 shows the concentrations of soluble TNF that were not blocked by soluble TNFR2 representing the biologically active form of the cytokine. Increased TNFR1-signaling in TNFR2^-/- BMDC differentiation cultures might occur.

BMDC sTNF kinetics

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 0.0

0.5 1.0 1.5

wt TNFR2

-/-sTNF [ng/mL]

Figure 27: sTNF concentrations in BMDC cultures - kinetics

At every time point of the kinetic 0.5 mL of the supernatant were removed from each petri dish of BMDC cultures and replaced by fresh GM-CSF-containing medium. The concentrations of soluble TNF were measured employing ELISA. Shown are the single values and the mean (horizontal line) of four different BMDC cultures per group representing individual mice.

3.3.1.7 TNFR2 concentrations in BMDC cultures

Furthermore, the concentrations of soluble TNFR2 were measured in the BMDC differentiation cultures, in order to acquire informations about its possible influence on the availability of biologically active soluble TNF. Wt BMDC cultures showed high concentrations of soluble TNFR2 in the supernatant after day 3 of the BMDC differentiation cultures period as demonstrated in Figure 28.

Figure 28: sTNFR2 concentrations in BMDC cultures - kinetics

At every time point of the kinetic 0.5 mL of the supernatant were removed from each petri dish of BMDC cultures and replaced by fresh GM-CSF-containing medium. The concentrations of soluble TNFR2 were measured employing ELISA. Shown are the single values and the mean (horizontal line) of four different BMDC cultures per group representing individual mice.