We read with interest the paper by Bonyhadi et al. (1) published February 15, 2005. We have, however, two concerns.

CD3/CD28 stimulation resulted in successful expansion of T cells from B-CLL PBMC samples. The authors state that this was associated with a rapid decrease in numbers of leukemic B cells. Given the average 1400-fold expansion of T cells seen, a drop in the percentage of B cells is not surprising, but this decrease may only be relative and not necessarily absolute as no actual cell numbers are given.

In addition, we have some misgivings about the apoptosis assay using a PE-conjugated anti-CD19 Ab in association with PI. After excitation with a 488-nm laser, PI fluoresces in the emission spectrums of both the FL2 and FL3 channel. In the system described, gating on the PE+ cells would therefore include all CD19+ cells and also all PI+ cells of any lineage, for example T cells. This would result in the apoptosis levels for “CD19+” cells appearing far greater than they actually are. An alternative approach, which we have used, is to use 7-AAD (which fluoresces in the FL3 channel only) as opposed to PI, allowing distinction between CD19+ cells and all other markers. Positively selected T cells from nine separate B-CLL PBMCs were activated overnight with CD3/28 beads (Dynal). These were subsequently cocultured with autologous B-CLL cells at ratios ranging from 1:1 to 1:5 T to B cells. Assessment of apoptosis of the CD19+ and CD19 populations was performed between 48 and 96 h of culture using FITC-annexin V and 7-AAD. In contrast to the results reported, we found that, in six of nine cases, viability was increased and apoptosis decreased in the CD19+ cells in cocultures compared with PBMCs alone (Table I). We do see a shift toward annexin positivity, but this is in the CD19 cells (representing T cells) from cocultures as opposed to the CD19+ cells (see Fig. 1).

Table I.

Increased viability and decreased apoptosis in CD19+ cells in cocultures compared with PBMCs alonea

% Viable Cells% Apoptosis
PBMCs aloneCoculturesPBMCs aloneCocultures
48 h     
 Case 1 25.3 35.9 74.2 63.3 
 Case 2 57.8 56.3 41.2 39.4 
     
72 h     
 Case 3 76.9 70.1 20.4 27.5 
 Case 4 13.5 33.0 85.7 65.3 
 Case 5 38.2 55.4 60.0 41.0 
 Case 6 69.0 46.5 29.8 51.8 
 Case 7 55.1 69.0 38.9 28.2 
     
96 h     
 Case 8 59.1 79.1 37.6 12.1 
 Case 9 89.2 92.8 9.4 6.3 
% Viable Cells% Apoptosis
PBMCs aloneCoculturesPBMCs aloneCocultures
48 h     
 Case 1 25.3 35.9 74.2 63.3 
 Case 2 57.8 56.3 41.2 39.4 
     
72 h     
 Case 3 76.9 70.1 20.4 27.5 
 Case 4 13.5 33.0 85.7 65.3 
 Case 5 38.2 55.4 60.0 41.0 
 Case 6 69.0 46.5 29.8 51.8 
 Case 7 55.1 69.0 38.9 28.2 
     
96 h     
 Case 8 59.1 79.1 37.6 12.1 
 Case 9 89.2 92.8 9.4 6.3 
a

Most cases show increased viability and decreased apoptosis of the CD19 fraction in cocultures compared to PBMCs alone. Only cases 3 and 6 show less viable cells and increased apoptosis in cocultures compared to PBMCs.

FIGURE 1.

Examples of two cases (upper panel, case 7; lower panel, case 1) using FITC-annexin V and 7-AAD to assess cell viability and apoptosis. In the cocultures, the CD19 fraction shows a shift to annexin positivity as opposed to the CD19+ fraction.

FIGURE 1.

Examples of two cases (upper panel, case 7; lower panel, case 1) using FITC-annexin V and 7-AAD to assess cell viability and apoptosis. In the cocultures, the CD19 fraction shows a shift to annexin positivity as opposed to the CD19+ fraction.

Close modal

In conclusion, we do not believe that CD3/CD28-activated T cells cultured with leukemic B cells in B-CLL necessarily results in increased apoptosis of malignant cells, and the increase seen in the work reported by Bonyhadi et al. may be the result of technical factors. Our data would suggest that the opposite may in fact be true.

We appreciate the comments by Drs. Patten, Devereux, and Buggins. It is likely that the differences in their results compared with ours are related to the experimental conditions utilized in their studies.

Dr. Patten et al. state that T cells were positively selected and activated overnight with the CD3/CD28 beads. We have examined the kinetics of activation of CLL patients’ T cells with CD3/CD28 beads in detail (R1 ). These studies have shown that induction of the key effector molecule CD154 (as well as others) is delayed compared with T cells from healthy donors. Several days of exposure of T cells from CLL patients to CD3/CD28 beads are required to activate and up-regulate key effector molecules. In our studies, we incubated the patients’ T cells with CD3/CD28 beads for 13 days. In contrast, Dr. Patten et al. reported a brief overnight incubation of the patients’ T cells with CD3/CD28 beads, which may be insufficient to achieve comparable levels of activation and effector molecule up-regulation. Second, if the beads were not removed following the short overnight incubation as described by Patten et al., they would remain firmly attached to the T cells and provide a physical barrier that could inhibit interaction of T cells with CLL cells, which we have shown in the paper is required for induction of apoptosis of the leukemic B cells. In our studies, a much longer incubation period (13 days) was utilized, which results in several hundred-fold increase in the total number of T cells in the cultures concomitant to the release of the beads from the T cells. In contrast to the experimental conditions of Patten et al., T cells are able to directly interact with CLL cells, thereby inducing CD95 up-regulation and facilitating induction of apoptosis.

With regard to the issue of T cells possibly being counted as B cells undergoing apoptosis due to use of propidium iodide, we too were concerned about this possibility. To rule out this possibility, we labeled the patients’ CLL cells with CFSE and subsequently gated on this population to ensure that no T cells (non-CFSE-labeled) would be included in our analyses. The numbers we obtained by this approach were similar to those reported in our paper, so we are confident that our conclusions are correct.

Finally, while not presented in our paper, we have also tracked expression of CD86 and CD95 on leukemic B cells in primary cultures in which CD3/CD28 beads were incubated with PBMC from CLL patients. Leukemic B cells disappeared rapidly between days 6 and 9 of culture. This occurred shortly after CD154 was induced on T cells and CD86 and CD95 induced on tumor cells. We presume leukemic B cells may be killed in a similar manner in vivo and plan to study this possibility in the future.

R1
Bonyhadi, M., M. Frohlich, A. Rasmussen, C. Ferrand, L. Grosmaire, E. Robinet, J. Leis, R. T. Maziarz, P. Tiberghien, R. J. Berenson.
2005
. In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphocytic leukemia.
J. Immunol.
174
:
2366
-2375.
1
Bonyhadi, M., M. Frohlich, A. Rasmussen, C. Ferrand, L. Grosmaire, E. Robinet, J. Leis, R. T. Maziarz, P. Tiberghien, R. J. Berenson.
2005
. In vitro engagement of CD3 and CD28 corrects T cell defects in chronic lymphocytic leukemia.
J. Immunol.
174
:
2366
-2375.