We read with interest the paper by Bonyhadi et al. (1) published February 15, 2005, and also the interesting letter by Patten et al. (2). We have also studied the effect of activated T cells on cocultured chronic lymphocytic leukemia (CLL) lymphocytes and our results agree with those reported by Patten et al.
The use of a ratiometric flow cytometry method for the enumeration of cells (3) clearly demonstrated that T cells activated with anti-CD3- and anti-CD28-coated microbeads can sustain the viability of autologous leukemic B cells even over long culture times (16 days). When only percentages are studied, a marked reduction in the percentage of B-CLL cells is observed, but this decrease is apparent, not real. This is shown in Fig. 1⇓, where our cell enumeration experiment clearly demonstrates that coculture with activated T lymphocytes maintains the number of viable B-CLL cells in culture. We conclude that, under certain experimental conditions, CD3/CD28 bead-activated T cells can prolong the survival of cocultured CLL cells.
As reported by Patten et al., our experiments were performed without washing the microbeads. However, we believe that Bonyhadi’s argument that these beads can “provide a physical barrier that could inhibit the interaction of T cells with CLLcells” may not hold since we have also shown that uncoated or CD3/CD28 beads neither induce nor inhibit B-CLL lysis, but CD23/CD28 microbeads even stimulate the specific lysis of autologous B cells by T cells (Fig. 2⇓).
CD3/CD28 bead-activated T cells prolong the in vitro survival of B-CLL cells. PBMCs of B-CLL patients were cultured for 7, 11, or 16 days with (⋄) or without (•) anti-CD3- and anti-CD28-coated microbeads (TCE). A, the percentage of leukemia B cells. B, the number of leukemia B cells per microliter over the kinetics of the culture. Data of one representative experiment of three are shown.
CD23/CD28 beads induce the lysis of B-CLL cells in coculture with autologous T lymphocytes. PBMCs of B-CLL patients were cultured for 24 h with microbeads coated by anti-CD28 and anti-CD23 or antiCD3. B cell specific lysis was assessed by flow cytometry using the vital dye 7AAD and anti-CD19 antibodies. Data of one representative experiment of three are shown.
- Copyright © 2005 by The American Association of Immunologists
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