EHEB

Cytokine Production by PBMC Incubated with EHEB Cells

The leukemic microenvironment containing several immune cells, mainly lymphocytes and macrophages capable of producing various molecules including inflammatory cytokines, plays an important role in the development and outcome of the disease. Direct contact of peripheral blood mononuclear cells (PBMC) incubated with Epstein-Barr virus (EBV)-transformed B-CLL cell line (EHEB) cells induced a marked increase of TNFα, IL-1β, IL-6, IFNγ, and IL-10 released by the immune cells.

Incubation of PBMC with an increasing number of EHEB cells caused a dose-dependent stimulation of TNFα release (F = 24.85, p < 0.001). There was no significant difference in TNFα production between PBMC (2 × 10⁶) incubated with 5 × 10⁴ or with 2 × 10⁵ of EHEB cells (Fig. 1). Incubation of PBMC with EHEB cells induced a stimulated IL-6 production (F = 12.27, p = 0.0016). In the presence of the three concentrations of EHEB cells, there was a threefold enhancement in IL-6 production (p < 0.05); however, no significant difference was found at the various concentrations (Fig. 1).

A dosed-dependent stimulation of IL-1β production was observed when PBMC (2 × 10⁶) were incubated for 24 hours with 1.25 × 10⁴ and 2 × 10⁵ EHEB cells (F = 77.7, p < 0.0001). The difference in IL-1β secretion in the presence of the two higher numbers of EHEB cells was not statistically significant (Fig. 2). The production of IFNγ by PBMC incubated with an increasing number of EHEB cells was significantly enhanced (F = 16.9, p = 0.0005) (Fig. 2). The secretion of IL-10 by PBMC was increased upon 24 hours of incubation with EHEB cell at the three concentrations tested (F = 47.6, p < 0.0001) (Fig. 3). There was no significant difference in the amount of IL-1ra produced by PBMC incubated for 24 hours without or with EHEB cells at concentration as indicated above (F = 0.64, p = 0.61) (Fig. 3).

Fig. 1 TNFα and IL-6 production by PBMC incubated for 24 hours with EHEB cells (cell-to-cell) or supernatants (Sup). (Bessler H, et al., 2020)

Fig. 2 IL-1β and IFNγ production by PBMC incubated for 24 hours with EHEB cells (cell-to-cell) or supernatants (Sup). (Bessler H, et al., 2020)

Fig. 3 IL-10 and IL-1ra production by PBMC incubated for 24 hours with EHEB cells (cell-to-cell) or supernatants (Sup). (Bessler H, et al., 2020)

Resistance to CdA of the Human B-cell Leukemia Cell Line EHEB

The effects of 2-chloro-2'-deoxyadenosine (CdA, cladribine), an adenosine deaminase-resistant analog toxic for both proliferating and resting lymphoid cells, were investigated in the human leukemia cell line EHEB, which was derived from a patient with B-CLL. The resistance of the human B-leukemia cell line EHEB to CdA does not result from an impairment of its phosphorylation into CdATP but from a complex series of events encompassing poor inhibition of ribonucleotide reductase and an increase in the proportion of DNA-synthesizing cells in S phase.

Sensitivity to CdA of the B-cell CLL cell line EHEB was evaluated by the MTT reduction assay after 4 days of incubation and compared with that of B-cell CLL lymphocytes freshly isolated from patients (Fig. 4). With an IC₅₀ of approximately 5 μm, EHEB cells appear to be 25-fold less sensitive to CdA than B-cell CLL lymphocytes, for which an IC₅₀ of 0.2 μm was calculated.

To evaluate ribonucleotide reductase activity in situ, the effect of CdA on the conversion of labeled Cyd into deoxynucleotides and its incorporation into DNA was analyzed. As depicted in Fig. 5, sizeable inhibition of the conversion of [³H] Cyd into dCTP and its incorporation into DNA was not observed at concentrations of CdA below 10 μm. An inhibition of ribonucleotide reductase in situ by CdA with an IC₅₀ of less than 0.1 μm in CCRF-CEM cells. Thus, inhibition of ribonucleotide reduction required more than 100-fold higher concentrations of CdA in EHEB cells than in CCRF-CEM cells.

Fig. 6 shows the scattergrams obtained by FACScan analysis of cells incubated for 24 h without (Fig. 6A) or with 10 μm CdA (Fig. 6B). The proportion of cells in the G1 phase was significantly decreased after a 24-h incubation with 10 μm CdA, whereas the proportion of cells synthesizing DNA (in either early or mid-late S phase) was significantly increased. CdA did not increase the rate of DNA synthesis but only increased the number of cells synthesizing DNA.

Fig. 4 Effect of CdA on viability of EHEB and B-cell CLL cells. (Cardoen S, et al., 2001)

Fig. 5 Dose effect of CdA on Cyd conversion into dCTP and its incorporation into DNA. (Cardoen S, et al., 2001)

Fig. 6 Simultaneous analysis of DNA synthesis and DNA content by BrdUrd incorporation, PI staining, and fluorescence-activated cell-sorting analysis. (Cardoen S, et al., 2001)