Phagocytosis assays with VISTA overexpressing macrophages engulfing

Macrophages are professional phagocytes which means that they are capable to engulf big pathogens, particles or apoptotic cells. To test the ability of phagocytosis is a standard test for functionality of macrophages. In this case the myeloid celllines HL-60 and THP-1 were differentiated into macrophages. The ability of phagocytosis was tested by phagocyting the B-cell lymphoma celllines SudHL4 or SudHL10 because these are known celllines which were recognized and engulfed easily and with a high percentage. In addition, 1 µg/mL of the antibody Rituximab against the B-lymphocyte surface molecule CD20 was added, to flag the B-lymphocytes for phagocytosis.

Figure: 4.3.3.1: Example experiment: phagocytosis of SudHL4 by HL-60 celllines (M0 macrophages), addition of 1 µg/mL Rituximab, n=1 in triplicates

The phagocytosis of SudHL4 by HL-60 cells showed a big difference after 2 hours already (Figure 4.3.3.1). HL-60 VISTA cells showed a much higher percentage of cells which engulfed SudHL4 cells compared to HL-60 EV cells. The difference was even stronger after 3-4 hours of phagocytosis up to more than 25 % difference. HL-60 EV

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cells did not have a high phagocytosis activity with a maximum of 12 %, which stayed constantly low. HL-60 VISTA-GFP cells however showed a much higher phagocytosis which increased up to 3 hours and remained high at around 31 %.

Figure 4.3.3.2: Pictures of phagocytosis of SudHL10 (red) by different macrophage types of HL-60 cells, addition of 1 µg/mL Rituximab

But the celllines could not only be differentiated into M0 macrophages, but also into other macrophage types (Figure 4.3.3.2). For all macrophage types we could observe much more red tumor cells engulfed by the VISTA overexpressing macrophages, but also more tumor cells which were attached to the macrophages. This could be due to the enhanced adhesion and promoted cell-cell contact of VISTA overexpressing celllines. Phagocytosis first needs attachment of phagocyte and tumor cell to occur.

So, the increased adhesion and cell-cell contact of VISTA overexpressing cells could also be a reason for the increased phagocytosis.

Figure 4.3.3.3: Example experiment: phagocytosis of SudHL10 for four hours by different macrophage types of HL-60 and THP-1 celllines, addition of 1 µg/mL Rituximab, n=2 in duplicates

When we quantified the percentage of macrophages, which performed phagocytosis, HL-60 VISTA-GFP cells (between 39-51 %) showed much higher phagocytosis compared to the control cells HL-60 EV (between 8,5-17 %). The phagocytosis of THP-1 was already quite high for wildtype cells (43,5-56 %), limiting the possibility of

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phagocytosis increase. But for all macrophage types, THP-1 VISTA-GFP cells also showed more phagocytosis (64-74 %) than the wildtype cells.

There was no clear difference observable for the different macrophage types. Normally you would expect the highest phagocytosis for M1 macrophages. M1 macrophages are typical killer macrophages with pro-inflammatory, bactericidal and phagocytic functions (Hesketh et al. 2017). M0 macrophages should have the second highest phagocytosis. M2 macrophages have a repair function in wound healing or tissue repair. Resident tissue macrophages are M2a type, while M2c macrophages turn off the damaging immune system (Galdiero et al. 2013). Because of their immunosuppressive function in the body, you would expect less phagocytosis of M2 macrophages. But former results with the wildtype celllines HL-60 and THP-1 of the Experimental Hematology at the UMCG showed the highest phagocytosis for M2c or M0 macrophage types. This is in accordance with my results.

Figure 4.3.3.4: Example experiment: number of engulfed tumor cells after 4 h phagocytosis of SudHL10 by M2c macrophages of HL-60 and THP-1 celllines, addition of 1 µg/mL Rituximab, n=2 in duplicates

VISTA-GFP overexpressing celllines did not only show more cells which did phagocytosis, but in addition they engulfed more tumor cells per phagocyte than the control cells (Figure 4.3.3.4). While 96 % of the phagocyting HL-60 EV cells engulfed only one tumor cell, HL-60 VISTA-GFP cells were more active. Only 67,4 % engulfed one tumor cell, 23,5 % already two tumor cells and 9 % even three or more tumor cells.

THP-1 wildtype cells engulfed comparable tumor cell numbers like HL-60 VISTA-GFP cells. But THP-1 VISTA-GFP cells also engulfed even more tumor cells/phagocyte than the wildtype. Only 45 % engulfed one tumor cell, 29 % two tumor cells and nearly 26

% engulfed three or more tumor cells. That showed that VISTA overexpression did not only lead to a higher percentage of macrophages performing phagocytosis, but also with a higher activity and more engulfed tumor cell per phagocyte.

Summarizing all three experiments of phagocytosis of SudHL10 cells by HL-60 and THP-1 macrophages (Figure 4.3.3.5) led to a high standard deviation. This was due to

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the individual activation state of the cells. For example, one aspect for the variability was the cell number. A defined number of cells was seeded, but dependent on their condition, the number of cells which differentiated and how strong they differentiated and attached differed slightly. With that, the cell-cell contact, and activity of the macrophages also differed.

But one could still see a much higher phagocytosis for HL-60 VISTA-GFP cells than for HL-60 EV cells. For THP-1 cells, the difference was not so pronounced in the single experiments (Figure 4.3.3.3), leading to a weaker increased phagocytosis for THP-1 VISTA-GFP cells compared to THP-1 wt cells. But you can still observe the trend.

Figure 4.3.3.5: Summary of phagocytosis of SudHL10 by different macrophage types of HL-60 and THP-1 celllines, macrophage type grouped, addition of THP-1 µg/mL Rituximab, n=4

Regarding the macrophage type difference for all three experiment of SudHL10 phagocytosis, there were still only small differences observable (Figure 4.3.3.6). But in accordance with the former results of the UMCG working group and the single experiment (Figure 4.3.3.3), M0 and M2c macrophages showed the highest percentage of phagocyting macrophages.

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Figure 4.3.3.6: Summary of phagocytosis of SudHL10 by different of HL-60 and THP-1 celllines, cellline grouped, addition of 1 µg/mL Rituximab, similar dataset like Figure 4.3.3.5, n=4

4.1.4 Phagocytosis assays of primary macrophages engulfing