Effect of different regulatory agents on the inhibitory activity of CD34+ cells
. | . | No. of responding/nonresponding cultures‡ . | . | Statistical significance§ . | . | . | |||
|---|---|---|---|---|---|---|---|---|---|
| Treatment* . | MLR cultures† . | Responding . | Nonresponding . | P value . | P range . | Significance . | |||
| Anti-HLA-1 | |||||||||
| A | 16 | 0 | ND | ND | ND | ||||
| B | 9 | 7 | 0.0004 | P < .001 | S | ||||
| A# | 16 | 0 | ND | ND | ND | ||||
| D | 15 | 1 | 0.3016 | P > .1 | NS | ||||
| Anti-IL-10 | |||||||||
| A | 16 | 0 | ND | ND | ND | ||||
| B | 7 | 9 | 5.7E-06 | P < .001 | S | ||||
| C | 16 | 0 | ND | ND | ND | ||||
| D | 3 | 13 | 8.3E-17 | P < .001 | S | ||||
| Anti-CD28∥ | |||||||||
| A | 37 | 11 | ND | ND | ND | ||||
| B | 9 | 39 | 3.9E-25 | P < .001 | S | ||||
| C | 30 | 2 | ND | ND | ND | ||||
| D | 5 | 43 | 1.4E-37 | P < .001 | S | ||||
| Anti-CD2 | |||||||||
| A | 6 | 10 | ND | ND | ND | ||||
| B | 2 | 14 | 0.0024 | .01 > P > .001 | S | ||||
| C | 10 | 6 | ND | ND | ND | ||||
| D | 1 | 15 | 1.4E-20 | P < .001 | S | ||||
| IL-2¶ | |||||||||
| A | 27 | 5 | ND | ND | ND | ||||
| B | 13 | 19 | 4.6E-07 | P < .001 | S | ||||
| C | 28 | 4 | ND | ND | ND | ||||
| D | 10 | 22 | 6.6E-12 | P < .001 | S | ||||
| IL-12¶ | |||||||||
| A | 12 | 20 | ND | ND | ND | ||||
| B | 3 | 29 | 4.8E-08 | P < .001 | S | ||||
| C | 31 | 0 | ND | ND | ND | ||||
| D | 7 | 25 | 3.8E-25 | P < .001 | S | ||||
. | . | No. of responding/nonresponding cultures‡ . | . | Statistical significance§ . | . | . | |||
|---|---|---|---|---|---|---|---|---|---|
| Treatment* . | MLR cultures† . | Responding . | Nonresponding . | P value . | P range . | Significance . | |||
| Anti-HLA-1 | |||||||||
| A | 16 | 0 | ND | ND | ND | ||||
| B | 9 | 7 | 0.0004 | P < .001 | S | ||||
| A# | 16 | 0 | ND | ND | ND | ||||
| D | 15 | 1 | 0.3016 | P > .1 | NS | ||||
| Anti-IL-10 | |||||||||
| A | 16 | 0 | ND | ND | ND | ||||
| B | 7 | 9 | 5.7E-06 | P < .001 | S | ||||
| C | 16 | 0 | ND | ND | ND | ||||
| D | 3 | 13 | 8.3E-17 | P < .001 | S | ||||
| Anti-CD28∥ | |||||||||
| A | 37 | 11 | ND | ND | ND | ||||
| B | 9 | 39 | 3.9E-25 | P < .001 | S | ||||
| C | 30 | 2 | ND | ND | ND | ||||
| D | 5 | 43 | 1.4E-37 | P < .001 | S | ||||
| Anti-CD2 | |||||||||
| A | 6 | 10 | ND | ND | ND | ||||
| B | 2 | 14 | 0.0024 | .01 > P > .001 | S | ||||
| C | 10 | 6 | ND | ND | ND | ||||
| D | 1 | 15 | 1.4E-20 | P < .001 | S | ||||
| IL-2¶ | |||||||||
| A | 27 | 5 | ND | ND | ND | ||||
| B | 13 | 19 | 4.6E-07 | P < .001 | S | ||||
| C | 28 | 4 | ND | ND | ND | ||||
| D | 10 | 22 | 6.6E-12 | P < .001 | S | ||||
| IL-12¶ | |||||||||
| A | 12 | 20 | ND | ND | ND | ||||
| B | 3 | 29 | 4.8E-08 | P < .001 | S | ||||
| C | 31 | 0 | ND | ND | ND | ||||
| D | 7 | 25 | 3.8E-25 | P < .001 | S | ||||
ND indicates not done; S, significant; and NS, not significant.
The tested regulatory agents were added at the initiation of the MLR to the culture medium, at their optimal concentration, according to the manufacturer's recommendation and according to preliminary titration experiments, except for the anti-HLA-1. This agent was preincubated with the CD34+ cells and washed prior to their addition to the MLR culture
MLR cultures in which responder cells were stimulated against allogeneic PBMCs from the CD34+ cells' donor, in the absence (A and C) or presence (B and D) of CD34+ cells, were established
The potential of different agents to reverse the inhibitory regulatory activity of CD34+ cells was evaluated by comparing the inhibition in the presence (C and D) and in the absence (A and B) of the specific agent. Briefly, a 5-day MLR was established in which the responder cells were then recultured for an additional 7 days under limiting dilution in microtiter plates. For each experiment, the number of positive and negative cultures, tested at the highest effector cell concentration (40 000 cells per well), are shown. Wells were scored positive for CTL activity when Cr release exceeded the mean spontaneous release value by at least 3 standard deviations of the mean. The regulatory activity of CD34+ cells was evaluated by their capacity to inhibit alloreactive CTL-p clones in the MLR to which they were added at a ratio of 0.5:1 CD34+/responder cell. The addition of CD34+ cells to MLR against third-party stimulators has not led to a significant inhibition (P > .1). Thus, in a total of 5 experiments carried out in the absence of CD34+ cells, 3 of 96 anti-third-party MLR culture wells were scored negative while 6 of 96 were scored negative in the presence of CD34+ cells
The results were statistically analyzed by the χ2 test
The results represent a total of 3 experiments
The results represent a total of 2 experiments
The role of anti-HLA-1 antibody was tested by incubating the antibody with CD34+ cells prior to their addition to the MLR. Therefore, for the statistical analysis of results in culture D we used as a reference the results of culture A