IL10 does not influence complement dependent cytotoxicity after Rituximab

3.4.1 IL10 does not influence complement dependent cytotoxicity after Rituximab treatment

The effect of endogenous IL10 on CDC has been examined. At first, different B cell lines were tested for their responsiveness to IL10 stimulation. Therefore, the B cell lines have been stimulated with recombinant human IL10 and the responsiveness to this stimulus has been visualized via immunoblot of phosphorylated STAT3 (data not shown). The cell lines Balm3, Ramos and Karpas422 responded to IL10 stimulation with phosphorylation of STAT3. In OCI Ly3 constitutive activation of STAT3 was detected. These four cell lines were therefore used for the following experiments.

Viability and proliferation of these cell lines have been examined, following treatment with different concentrations of Rituximab, representing the induction of CDC. The lower the viability and the proliferation of the cells, the higher was the induced CDC.

CDC needs complement factors, which are usually destroyed via heat decomplementation of the serum used for cell culture medium. For this experiment human serum was used either heat decomplemented as a negative control or left untreated to retain complement factors. Before the treatment with Rituximab and complement, the cell lines were treated with IL10 for 3h. However, in none of the examined cell lines an influence of IL10 treatment could be observed (Figure 3-26).

The cell line Balm3 was lysed after treatment with complement and 10µg/ml Rituximab, without a difference between those cells that have been pretreated with IL10 and those without this treatment. Already the addition of complement seemed to reduce proliferation of the cells. Ramos cells reacted similar to Balm3. But for Ramos the addition of 0.1µ/ml Rituximab already reduced the proliferation considerably. In contrast to Balm3 and Ramos, Karpas422 and OCI Ly3 do not show a reduction of viability or proliferation upon Rituximab treatment. For OCI Ly3 a reduction of proliferation was observed after the addition of complement as it was seen for Balm3.

For both cell lines an enhanced viability could be seen after the addition of complement. However, this effect has not been observed in other experiments and is therefore not representative.

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Figure 3-26 No Influence of exogenous IL10 on complement dependent cytotoxicity after Rituximab treatment

Viability (left) and proliferation (right) of Balm3, Ramos, Karpas 422 and OCI Ly3 cell lines were measured after Rituximab treatment for 24h as a read-out for CDC. Cells were cultured either with decomplemented human serum (-complement) or with undecomplemented human serum (+complement) with (+IL10) or without (-IL10) preincubation with IL10 for 3h. Viability and proliferation of cells cultured without Rituximab and without complement are set to 100%. Rituximab was used in

Next, the effect of endogenous IL10 expression levels has been assessed. As seen in Figure 3-14, only the B cell lines MC116, Balm3 and BJAB express IL10 in detectable amounts. These cell lines have been analysed in comparison to cell lines without IL10 expression. All cell lines have been treated with different concentrations of Rituximab, with or without complement. Viability and proliferation have been measured as a read out for CDC. For some cell lines, namely MC116 and Ramos, lysis of the cells could be seen visually after treatment with 10µg/ml Rituximab on the 94-well plates used for the assays. As seen in Figure 3-27 for proliferation and Figure 3-28 for viability no influence of IL10 transcription could be observed for CDC. The addition of complement already leads to a reduction of proliferation in all examined cell lines. All three cell lines with IL10 expression showed a reduction of viability (Figure 3-28) after Rituximab treatment and a reduction of proliferation was observed for MC116 and Balm3 (Figure 3-27). For those cell lines without endogenous IL10 expression different results from complete lysis in Ramos and SuDHL4, represented by reduction in proliferation and viability, to no effect at all as seen for Karpas422 and OCI Ly3 have been observed. If endogenous IL10 expression would influence CDC, it would have been expected that cells from the one group would show stronger reaction on Rituximab treatment than cells from the other group. This has not been observed. Therefore, one can conclude that endogenous IL10 expression does not influence Rituximab-mediated CDC.

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Figure 3-27 Endogenous IL10 does not influence complement dependent cytotoxicity after Rituximab treatment, visualized by proliferation of cell lines.

Proliferation of cell lines with IL10 expression (MC116, Balm3, BJAB; upper lane) and cell lines without IL10 expression (Ramos, Bl2, Karpas422, SuDHL4 OCI Ly3 and OCI Ly1) was measured after Rituximab treatment for 24h as a read-out for CDC. Cells were cultured either with decomplemented human serum (-complement) or with undecomplemented human serum (+complement). Rituximab was used in three different concentrations (0.001µg/ml, 0.1µg/ml and 10µg/ml). Proliferation of cells cultured without Rituximab and without complement was set to 100%. The cell lines are ordered according to IL10 expression level. MC116 is the cell line with the highest IL10 expression level.

MC116

Figure 3-28 Endogenous IL10 does not influence complement dependent cytotoxicity after Rituximab treatment, visualized by viability of cell lines.

Viability of cell lines with IL10 expression (MC116, Balm3, BJAB; upper lane) and cell lines without IL10 expression (Ramos, Bl2, Karpas422, SuDHL4 OCI Ly3 and OCI Ly1) was measured after Rituximab treatment for 24h as a read-out for CDC. Cells were cultured either with decomplemented human serum (-complement) or with undecomplemented human serum (+complement). Rituximab was used in three different concentrations (0.001µg/ml, 0.1µg/ml and 10µg/ml). Proliferation of cells cultured without Rituximab and without complement was set to 100%. The cell lines are ordered according to IL10 expression level. MC116 is the cell line with the highest IL10 expression level.

11 7 Taken together, it has been shown that neither exogenous IL10 nor IL10 expression

levels influence Rituximab-mediated CDC.