Addition of rituximab to PMBCs leads to B cell depletion in the absence of serum
Freshly isolated PBMCs from 14 healthy donors were cultured with or without rituximab overnight. In all donors we observed a strong rituximab-mediated reduction in B cell numbers, and no B cells were detectable after rituximab treatment (<0.55 % of lymphocytes) in 10/14 donors (Fig. 1a). In the first experiments, we used anti-TNF alpha antibody infliximab or IvIgs as negative controls. We discontinued these controls in further experiments, as no effects on either the presence of B cells (Fig. 1a; infliximab, n = 2; IvIg, n = 1) nor the degree of NK cell degranulation was seen (see subsequent text; infliximab, n = 3; IvIg, n = 2). Infliximab had no effect on B cell proportions, even after culture over 4 days and contrary to rituximab (n = 2, not shown). B cell depletion was incomplete in 4/14 donors following rituximab treatment overnight (Fig. 1b, c). The expression of CD19 on the remaining B cells was decreased and viability staining with Annexin V revealed that an important fraction of these cells was apoptotic (Fig. 1b). Donors with incomplete B cell depletion had a significantly lower ratio of NK cells to B cells at baseline than donors with complete B cell depletion (Fig. 1c).
These data indicate that rituximab can induce B cell depletion in PBMCs without the presence of functional serum factors. Low ratios of NK cells to B cells might be responsible for incomplete, presumably delayed, B cell depletion.
Rituximab leads to NK cell degranulation and downregulation of CD16 in PBMCs
The degranulation of NK cells was measured in six donors after culture of freshly isolated PBMCs with or without rituximab (or control antibody infliximab, n = 3, and IvIg, n = 2) overnight. CD107a expression as a correlate of degranulation was increased only if rituximab had been added (Fig. 2a, b). In the six donors investigated, CD107a expression was statistically significantly higher in samples that contained rituximab than in samples that contained no therapeutic antibody (Wilcoxon signed rank test, p = 0.03; not shown).
The Fc-gamma-receptor CD16 was downregulated on degranulated (CD107a-positive) NK cells, as shown in Fig. 2c. The proportion of CD16bright cells among CD56dim NK cells was determined after culture with or without rituximab in 16 healthy controls. Rituximab led to a significant decrease in CD16bright NK cells (Fig. 2d). The extent of CD16 downregulation varied between donors.
We conclude that rituximab induces NK cell degranulation in healthy PBMCs. Similar to published data in tumor models, rituximab induced downregulation of CD16.
NK cells and serum cooperate in mediating rituximab-induced B cell depletion
To investigate a causal relationship between NK cell degranulation and the depletion of B cells upon rituximab treatment we depleted NK cells from freshly isolated PBMCs using anti-CD56 and anti-CD16 antibodies and magnetic beads. The remaining PBMCs were cultured overnight with or without rituximab and with or without autologous human serum.
Rituximab-induced B cell depletion was abrogated if NK cells were depleted from the PBMCs (Fig. 3a, n = 4 donors). However, the addition of active autologous human serum in addition to rituximab led to reduced numbers of B cells in NK-depleted PBMCs (Fig. 3b, n = 4). This effect was abrogated if the serum was heat inactivated, or if serum was not added (Fig. 3b). The greatest reduction in B cells was in samples containing both NK cells and active serum (Fig. 3c). The complete experiment with all negative controls, including the proof of successful NK cell depletion is shown in Additional file 1: Figure S1(a).
These data demonstrate that in the absence of serum, NK cells are responsible for rituximab-induced B cell depletion. In the absence of NK cells, serum factors such as complement alone can mediate rituximab-induced B cell reduction and NK cells and serum factors have a complementary effect on rituximab-induced B cell reduction.
The extent of rituximab-induced B cell depletion correlates with the size of NK cell proportions
To investigate the correlation between the amount of B cell depletion and the ratio of NK to B cells (Fig. 1), we resubstituted NK-cell-depleted PBMCs with autologous NK cells from three different healthy donors (Fig. 4a, b, and c, respectively). The addition of increasing amounts of NK cells led to increasingly profound decrease in B cells (Fig. 4, gray bars). This decrease was not an indirect consequence of increased NK cell proportions, as shown by the negative control samples cultured without rituximab (Fig. 4, black bars). One complete experiment with all combination scenarios (+/− rituximab, +/− NK cell depletion, +/− NK cell re-substitution) is shown in Additional file 1: Figure S1(b).
In order to statistically analyze this effect, the ratio (B cells (% of lymphocytes) in samples treated with rituximab)/(B cells in samples treated without rituximab) was calculated and related to the respective NK cell percentages (% of lymphocytes). The linear regression analysis of the donor shown in Fig. 4a was significant (R
2 = 0.9993, p = 0.0003), whereas the analysis of the donors shown in Fig. 4b and c were not significant (most likely due to limited measurements per donor). The pooled correlation analysis was significant (Spearman's r = −0.736, p = 0.0047). We conclude that B cell depletion can be accelerated by elevated NK cell proportions.
Rituximab leads to further phenotypic changes in CD56dim NK-cells
We investigated the expression of the lymphocyte activation marker CD69 on CD56dim NK cells from freshly isolated PBMCs from 13 healthy donors. After culture with rituximab overnight, CD69 expression was statistically significantly greater than after culture without rituximab (Wilcoxon signed rank test, p = 0.0002; Fig. 5a). A median of 25 % of CD56dim NK cells expressed CD69 de novo.
Using the same culture conditions, the expression levels of the NK cell co-activation receptor CD137 (41BB) was investigated in 11 donors (Fig. 5b). Rituximab significantly increased the expression of CD137 (p = 0.001). A median of about 10 % of CD56dim NK cells expressed CD137 as a response to rituximab.
Therefore, next to the effects of rituximab that can be directly assigned to Fc-γ-receptor CD16 signaling (such as degranulation and downregulation of CD16), an important fraction of CD56dim NK cells showed unspecific signs of activation and the expression of at least one co-activating receptor was altered.
NK cells exposed to rituximab can become hypo-responsive in a donor-dependent fashion
We hypothesized that the phenotypic changes described might impact the NK cell cytotoxicity towards other target cells. To explore this, we performed a series of killing assays with 11 healthy donors. Freshly isolated PBMCs were cultured overnight with or without rituximab, washed, and then co-cultured for 4 hours with the NK cell-sensitive, 51Cr-labeled K562 cell line. Lysis of K562 cells by NK cells is independent of Fc-receptors.
We observed donor-dependent effects of rituximab on NK cell cytotoxicity. While many donors had no reduction or only slight reduction in cytotoxicity after culture with rituximab (Fig. 6a) we observed statistically significant reduction in NK cell cytotoxicity upon rituximab pretreatment in three donors (Fig. 6b). Due to this donor variability, on statistical analysis of the overall NK cell cytotoxicity in all donors there was a non-significant trend towards reduced NK cell cytotoxicity upon culture with rituximab (Fig. 6c).
These data indicate that incubation of non-malignant PBMCs with rituximab alters NK cell cytotoxicity in a donor-dependent fashion.