To the Editor:

We have been much interested in the recent report by Mielcarek et al1 that proposed one of the mechanisms explaining the unexpectedly low incidence of acute graft-versus-host disease (GVHD) after allogeneic peripheral blood stem cell transplantation (PBSCT) despite large numbers of T cells infused. They showed that T-cell proliferative responses to alloantigen stimulation were suppressed after granulocyte colony-stimulating factor (G-CSF) administration by a large number of monocytes in apheresis products. We also investigated alloantigen-stimulated immune responses of G-CSF–mobilized peripheral blood mononuclear cells (PBMNCs) from normal donors using a mixed lymphocyte culture system. PBMNCs before G-CSF administration (pre–G-CSF) and samples from leukapheresis (post–G-CSF) were obtained from six donors. Adherent cells were roughly removed by the plastic adherence at 37°C for 1 hour. The remaining cells (0.5 or 1 × 105 cells) were cultured with an equal number of irradiated (30 Gy) allogeneic PBMNCs in 200 μL of RPMI 1640 supplemented with 10% pooled human AB serum for 7 days in a flat-bottomed 96-well plate. For the last 18 hours, cultures were pulsed with 3H-thymidine (0.25 μCi/well). Cells were harvested on glass fiber filters and 3H-thymidine uptake was measured by liquid scintillation counting. Pre–G-CSF PBMNCs showed significantly lower proliferative responses compared with post–G-CSF PBMNCs in four of six donors, whereas the response was equivalent between pre–G-CSF and post–G-CSF PBMNCs in the remaining two donors (Table 1). This interindividual variation is consistent with the observation by Mielcarek et al.1 

Table 1.

Proliferative Response of Pre–G-CSF and Post–G-CSF PBMNCs to Alloantigen Stimulation

DonorCells3H-Uptake (cpm)
Pre–G-CSFPost–G-CSF
MNC* 4,968 (633) 265 (14) 
MNC* 4,419 (328) 205 (53) 
MNC* 2,212 (328) 2,668 (197) 
MNC 6,648 (1,845) 8,287 (934) 
MNC 10,324 (506) 706 (78) 
MNC 13,157 (3,064) 866 (193) 
 CD4+ 8,611 (1,936) 9,957 (52) 
 CD8+ρ 894 (246) 1,382 (181) 
DonorCells3H-Uptake (cpm)
Pre–G-CSFPost–G-CSF
MNC* 4,968 (633) 265 (14) 
MNC* 4,419 (328) 205 (53) 
MNC* 2,212 (328) 2,668 (197) 
MNC 6,648 (1,845) 8,287 (934) 
MNC 10,324 (506) 706 (78) 
MNC 13,157 (3,064) 866 (193) 
 CD4+ 8,611 (1,936) 9,957 (52) 
 CD8+ρ 894 (246) 1,382 (181) 

Pre–G-CSF and post–G-CSF PBMNCs or purified CD4+ or CD8+ cells were cultured with the same number of irradiated allogeneic PBMNCs for 7 days. Data are expressed as a mean (SD) of triplicates.

*

5 × 104 cells.

1 × 105 cells.

4 × 104 cells.

ρ 2 × 104 cells.

Post–G-CSF PBMNCs contained a larger number of monocytes than did pre–G-CSF PBMNCs, as already pointed out.1,2 We then evaluated allogeneic immune responses of purified CD4+ or CD8+ cells from a donor in whom the allogeneic response of post–G-CSF PBMNCs was markedly diminished. CD4+ and CD8+ cells were selected from PBMNCs by using Mini-MACS (Miltenyi Biotec Inc, Sunnyvale, CA) according to the manufacturer's protocol. Briefly, PBMNCs were incubated with mouse IgG1 antihuman CD4 or CD8 antibodies for 30 minites at 4°C, washed twice, and incubated with antimouse IgG microbeads for 30 minutes at 4°C. CD4+ or CD8+ cells were then separated from the negative selection using magnetic column and separator. Purified CD4+ (4 × 104) or CD8+ cells (2 × 104) were cultured with an equal number of irradiated stimulator cells and 3H-thymidine uptake was measured as described above. The responses of post–G-CSF CD4+ or CD8+ cells were similar to that of the pre–G-CSF corresponding cells. Furthermore, we investigated in vitro effects of G-CSF on proliferative response of T cells to alloantigen stimulation using PBMNCs from four volunteers. G-CSF did not affect proliferation induced by alloantigen in vitro (Table 2). These results suggest that G-CSF does not directly affect the proliferative response of T cells to alloantigen stimulation both in vivo and in vitro. The hyporesponsiveness of post–G-CSF PBMNCs could be due to accessary cells other than T cells contained in apheresis products. To confirm this hypothesis, we investigated whether monocytes could suppress proliferative responses of alloantigen-stimulated T cells in vitro. After the incubation of PBMNCs obtained from a normal volunteer in a culture flask coated with fetal calf serum (FCS) at 37°C for 12 hours, nonadherent cells were removed, washed twice, and resuspended in RPMI supplemented with 10% AB serum. Adherent cells were gently removed with phosphate-buffered saline containing 5% FCS and 0.5% ethylenediaminetetraacetic acid. Cultures were established with 1 × 105 nonadherent PBMNCs and an equal number of irradiated allogeneic PBMNCs in the presence of different numbers of autologous adherent cells and different concentrations of G-CSF. Figure 1 clearly shows that the monocytes suppress the proliferative response of T cells in a dose-dependent manner, and the presence of G-CSF in the culture did not show any effects on T-cell responses. This is consistent with the result reported by Mielcarek et al1 that shows that the suppressive effect of post–G-CSF CD14+ monocytes was not different from that of pre–G-CSF monocytes. Taken together, the suppression of acute GVHD induced by donor T cells could be best explained by the quantitative effect of monocytes in allografts. However, it was shown that cultures of PBMNCs in the presence of G-CSF led to decreased production of tumor necrosis factor-α.3 Mielcarek et al1 showed downexpression of B7-2 on monocytes after G-CSF administration. These in vivo and in vitro data suggest that G-CSF could alter monocyte functions. In mice, G-CSF treatment was found to polarize donor T cells toward a type-2 T-helper response with decreased interleukin-2 and interferon-γ production and corresponding reduction in acute GVHD.4 G-CSF effects on T cells and monocytes should be further investigated in terms of cytokine-producing ability and surface molecule expression, because differences in composition of the allografts between peripheral blood and bone marrow have the potential to alter the incidence and severity of GVHD, the frequency of graft rejection, the kinetics of hemopoietic the engraftment and immune reconstitution, and the magnitude of graft-versus-leukemia effects.

Table 2.

In Vitro Effects of G-CSF on Proliferative Response of PBMNCs to Alloantigen Stimulation

Experiment No.G-CSF (ng/mL)
021050
3,841 (52) 3,546 (920) 4,967 (566) 3,759 (632) 
7,368 (1,499) 9,032 (2,915) 9,913 (1,652) 8,460 (400) 
4,861 (667) 5,528 (570) 5,603 (1,205) 6,325 (1,036) 
6,785 (1,767) 6,758 (1,010) 7,168 (446) 7,643 (1,399) 
Experiment No.G-CSF (ng/mL)
021050
3,841 (52) 3,546 (920) 4,967 (566) 3,759 (632) 
7,368 (1,499) 9,032 (2,915) 9,913 (1,652) 8,460 (400) 
4,861 (667) 5,528 (570) 5,603 (1,205) 6,325 (1,036) 
6,785 (1,767) 6,758 (1,010) 7,168 (446) 7,643 (1,399) 

Values are 3H-uptake (cpm). PBMNCs (1 × 105 cells) were cultured with the same number of irradiated allogeneic PBMNCs for 7 days in the absence or presence of various concentrations of G-CSF. Data are expressed as a mean (SD) of triplicates.

Fig. 1.

Nonadherent PBMNCs (1 × 105) and an equal number of irradiated allogeneic PBMNCs were cultured in the presence or absence of various numbers of autologous adherent cells and various concentrations of G-CSF. Data are expressed as the mean and standard deviation.

Fig. 1.

Nonadherent PBMNCs (1 × 105) and an equal number of irradiated allogeneic PBMNCs were cultured in the presence or absence of various numbers of autologous adherent cells and various concentrations of G-CSF. Data are expressed as the mean and standard deviation.

ACKNOWLEDGMENT

Supported in part by grants-in-aid from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture (06454348), and Uehara Memorial Foundation.

REFERENCES

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