Mixed and non-stimulated BMDC cultures

In order to address whether different culture conditions in terms of soluble TNF and soluble TNFR2 influence the phenotypes seen in TNFR2-/- BMDC cultures, both populations were differentiated in the same petri dish, in order to guarantee identical conditions. Mixed BMDC cultures were obtained by preparing bone marrow cells from one wildype and one TNFR2 -/-mouse and mixing equal numbers of these cells. Hence, the resulting mixed culture should consist of 50% wt and 50% TNFR2^-/- bone marrow cells. These cultures were differentiated into BMDC. At the indicated time points the two populations in the mixed BMDC cultures were investigated using flow cytometry or FACS Aria cell sort device.

3.3.3.1 Cell proportions in mixed BMDC cultures

In order to find out, whether the reduced yield of BMDC of TNFR2^-/- mice is due to missing intrinsic signaling or depending on altered culture conditions, the frequencies of the respective population in mixed BMDC differentiation cultures were analyzed at different time points. Figure 33 illustrates that TNFR2^-/- BMDC were present in an above-average frequency in mixed cultures on day 3. In contrast to this, the percentage of TNFR2^-/- BMDC decreased steadily and reached significantly lower levels compared to the wt BMDC on day 8 and day 10. These

findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 23.

Figure 33: BMDC distribution in mixed cultures - kinetics

Mixed BMDC were analyzed for the distribution of CD45.1 wt and CD45.2 TNFR2^-/- cells at the time points indicated using flow cytometry. Shown are the single values and the mean (horizontal line) of four different mixed BMDC cultures per group representing individual mice.

3.3.3.2 Frequency of cells expressing activation markers (MHCII

CD80

CD86

) in mixed BMDC cultures

Afterwards, the influence of TNFR2^-/- BMDC culture conditions on the activation of the developing BMDC should be figured out. Therefore the frequencies of activated cells in the two different populations of mixed BMDC differentiation cultures were examined at different time points. The relative proportions of activated cells in the TNFR2^-/- BMDC population of mixed BMDC cultures started lower on day 4 and day 6 but were significantly increased on day 10.

Data are interpreted in Figure 34. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 24.

BMDC distribution kinetics

Day 4 Day 6 Day 8 Day 10 0

20 40 60 80

wt TNFR2

-/-p=0.0027 p=0.0004 p<0.0001

[% congenic live cells]

BMDC activation kinetics

Day 4 Day 6 Day 8 Day 10 0

5 10 15 20 25

wt TNFR2

-/-p=0.0004

CD80+ CD86+ MHCII+ [% live cells]

Figure 34: Activation markers expression in mixed BMDC cultures - kinetics

Mixed BMDC were analyzed for the percentages of activated cells (MHCII⁺ CD80⁺ CD86⁺) in the two populations that were distinguished by their congenic markers. Measurements were done at the time points indicated using flow cytometry. Shown arethe 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.

3.3.3.3 Frequency of MDSC in mixed BMDC cultures

Moreover, the frequencies of cells expressing the markers of MDSC (CD11b⁺ Ly6C⁺ Ly6G^-) in the two different populations of mixed BMDC differentiation cultures were examined at different time points. The relative proportions of MDSC in the TNFR2^-/- population of mixed cultures were significantly decreased after day 4. Data are shown in Figure 35. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 19.

A

B

Figure 35: MDSC in mixed BMDC cultures - kinetics

Mixed BMDC were analyzed for the percentages of MDSC (CD11b⁺ Ly6C⁺ Ly6G^-) in the two BMDC populations that were distinguished by their congenic markers. Measurements were done at the time points indicated using flow cytometry. Shown are the single values and the mean (horizontal line) of four different mixed BMDC cultures per group representing individual mice.

3.3.3.4 Proliferation of mixed BMDC cultures

The proliferation was measured in the two populations of mixed BMDC differentiation cultures.

The relative proportions of proliferating cells in the TNFR2^-/- population of mixed cultures were slightly decreased on day 10. The data are shown in Figure 36. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 25.

Figure 36: Proliferation in mixed BMDC cultures

Mixed BMDC were analyzed for the percentage of proliferating cells (anti-BrdU FITC) in the two BMDC populations that were distinguished by their congenic markers. Measurements were performed on day 10 using flow cytometry. Cells were incubated with BrdU for the 24 previous hours. Shown are the single values and the mean (horizontal line) of three different mixed BMDC cultures per group representing individual mice.

BMDC proliferation d10

wt

TNFR2-/-0 5 10 15

BrdU+ [% congenic live cells]

BMDC MDSC kinetics

Day 4 Day 6 Day 8 Day 10 0

20 40 60 80

wt TNFR2

-/-p=0.0002 -/-p=0.0002 p=0.0001

MDSC [% live cells]

3.3.3.5 Cell death in mixed BMDC cultures

In parallel, the frequencies of dead cells in the two populations of mixed BMDC differentiation cultures were analyzed. The relative proportions of dead cells in the TNFR2^-/- population of mixed cultures were comparable to the percentages in the wt population. Data are illustrated in Figure 37. These findings correspond to the results from BMDC generated in separated BMDC differentiation cultures shown in Figure 26.

Figure 37: Cell death in mixed BMDC cultures

Mixed BMDC were analyzed for the percentages of apoptotic and necrotic cells (Annexin V⁺ 7-AAD⁺) in the two BMDC populations that were distinguished by their congenic markers. Measurements were performed on day 10 using flow cytometry. Shown are the single values and the mean (horizontal line) of three different mixed BMDC cultures per group representing individual mice.