Despite a 10-fold increase of T cell dose, the incidence and severity of acute GVHD following allogeneic transplantation of G-CSF-mobilized PBSC is not increased compared to BMT. Experimental murine studies demonstrate that G-CSF polarizes donor T cells toward a type 2 cytokine response. To determine whether G-CSF alters T cell cytokine responses, we investigated the effects of G-CSF administration on T cell proliferative and cytokine responses to alloantigen and Con A in nonadherent PBMC (NAC) and CD3+ T cells obtained from normal individuals before and after G-CSF administration (10 μg/kg × 4 days). Although T cell proliferative and cytokine (IFN-γ and IL-4) responses to alloantigen stimulation and Con A were significantly reduced in post-G-CSF NAC, they were restored by the removal of non-T cells from post-G-CSF NAC. Furthermore, there was less T cell alloreactivity in MLR in the presence of autologous post-G-CSF monocytes than in the presence of pre-G-CSF monocytes. This alteration was not replicated in vitro by culturing PBMC with G-CSF. These results suggest that G-CSF administration suppresses T cell proliferative and cytokine (IFN-γ and IL-4) responses to allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplantation (2000) 25, 1035–1040.
G-CSF has been shown to expand the PBSC pool, and G-CSF-mobilized PBSC from HLA-matched siblings are increasingly being used for allogeneic transplantation (PBSCT).12345 Recent clinical evidence has shown that allogeneic PBSCT results in faster hematologic engraftment and immunologic reconstitution than allogeneic BMT,678 perhaps due to the large number of hematopoietic progenitor cells and mature lymphocytes in PBSC products. Interestingly, comparison of G-CSF-mobilized PBSCT and BMT demonstrates a similar incidence and severity of acute GVHD.5 This is surprising because leukapheresed PBSC grafts contain at least a one log greater number of T cells than is present in bone marrow harvests.12345 The relative reduction of acute GVHD may be attributed to immunomodulatory effects of G-CSF on donor T cells. In experimental murine models of allogeneic PBSCT, G-CSF treatment in donors has been found to polarize donor T cells towards a type 2 cytokine response with increased IL-4 production and decreased IL-2 and IFN-γ production, corresponding to a reduction in acute GVHD.91011 Polarized type 2 alloreactive donor T cells have been shown to protect mice from acute GVHD.12 However, type 2 polarization by G-CSF has yet to be demonstrated in humans. Instead, studies of G-CSF-mobilized blood in humans have demonstrated a reduced T cell proliferative response of PBMC in MLR after G-CSF administration.1314151617181920 This suppression has been attributed to large amounts of monocytes in G-CSF-mobilized PBSC.15162021 In order to address these issues, we studied proliferative and cytokine responses to alloantigens and Con A of nonadherent PBMC (NAC) and CD3+ T cells positively selected from NAC obtained from normal individuals before and after G-CSF administration.
Materials and methods
Seven donors for allogeneic PBSCT and four normal volunteers entered into the study after giving informed consent to this protocol, which was approved by the Institutional Review Board of Okayama University Hospital. Ages of donors and volunteers ranged from 20 to 42 years. Seven were male and two were female. Each individual was treated subcutaneously with recombinant human G-CSF (Kirin-Sankyo, Tokyo, Japan; Chugai Pharmaceuticals, Tokyo, Japan; and Kyowa Pharmaceuticals, Tokyo, Japan) at a dose of 10 μg/kg once daily for 4 or 5 days.
Heparinized peripheral blood samples were obtained by venous puncture or leukapheresis (LP) before and after G-CSF administration. LP was performed using a Cobe Spectra Cell Separator (COBE Laboratories, Lakewood, CO, USA) as described previously.22 The LP products were centrifuged at 200 g for 5 min to remove platelets. PBMC were isolated from the heparinized blood or the LP products by Histopaque (1.077 g/ml, Sigma, St Louis, MO, USA) density gradient centrifugation, washed twice in phosphate-buffered saline (PBS) and resuspended in RPMI 1640 supplemented with 10% pooled, heat-inactivated human AB serum, 100 U/ml penicillin and 100 μg/ml streptomycin (complete medium). PBMC were then incubated in a plastic dish at a concentration of 2 × 106 cells/ml at 37°C for 60 min to remove plastic-adherent cells. Nonadherent PBMC obtained before and after G-CSF administration were referred to as preG-NAC and G-NAC, respectively. Remaining adherent cells were incubated in PBS supplemented with 0.5% ethylenediaminetetraacetic acid (EDTA) and fetal calf serum (FCS) for 30 min at 4°C during which time they detached from the T-75 flask (Coaster, Cambridge, MA, USA) surface. The supernatant containing adherent cells was then harvested, washed three times with RPMI 1640 containing 10% FCS, and resuspended in complete medium.
CD3+ cells were positively isolated using Dynabeads M-450 CD3 MoAb (Dynal, Oslo, Norway) according to the manufacturer's protocols. Briefly, cells were stained with CD3 beads at a 1:4 ratio and incubated for 30 min at 4°C on an apparatus that provides both gentle tilting and rotation. After incubation, rosetted cells positively selected using a magnet were incubated overnight in complete medium at 37°C in 5% CO2 in T-75 flasks to remove the beads from cells. CD3+ cells were then collected after removing M-450 CD3 beads with a magnet. According to the manufacturer's protocol, CD3 antigen expression, which is transiently downregulated during these procedures, is recovered on the cell surface after 24 h of culture.
In some experiments CD4+ cells were also positively selected after primary MLR by using Mini-MACS (Miltenyi Biotec, Sunnyvale, CA, USA) according to the manufacturer's protocol. Briefly, cells were incubated with mouse IgG1 anti-human CD4 MoAb at 4°C for 30 min. CD4+ cells were then separated by positive selection using a magnetic column and separator. Cells were resuspended in complete medium for further experiments. Purity of the CD3+ cells and CD4+ cells was always more than 90% and the viability of these cells exceeded 95% as determined by trypan blue exclusion.
Flow cytometric analysis
Surface marker analyses were performed by one- or two-color flow cytometry using a FACS Calliber (Becton Dickinson, San Jose, CA, USA). All MoAb were purchased from Becton Dickinson. Cells were stained with MoAb conjugated with PE directed to CD8 and CD14, or with MoAb conjugated with FITC directed to CD3 and CD4. Conjugated isotype-matched antibodies were used for control staining. Sample suspensions were adjusted to 106 cells/ml and incubated on ice with the appropriate antibody for 20 min, washed twice, and were resuspended in 0.5 ml 1% bovine serum albumin (BSA) in PBS. Dead cells were excluded by electronic gating and 30 000 gating events were analyzed for each sample.
Analysis of cell proliferation
Responder cells (1 × 105 cells) were cocultured with irradiated (30 Gy), third party allogeneic stimulator cells (PBMC; 1 × 105 cells) for 7 days or with 10 μg/ml of Con A (Vector Laboratories, Burlingame, CA, USA) for 3 days in 200 μl of complete medium in a 96-well, flat-bottomed plate (Costar) at 37°C in 5% CO2 in triplicate. The cultures were then pulsed with 0.25 μCi per well of 3H-thymidine for the last 18 h of culture. Cells were harvested and 3H-thymidine incorporation was measured by liquid scintillation counting. In some experiments, 1 × 105 of pre-G-CSF PBMC were cultured with 1 × 105 irradiated allogeneic PBMC in triplicate for 7 days in the absence or presence of various numbers of autologous pre-G-CSF or post-G-CSF adherent cells.
In order to determine the in vitro effects of G-CSF on T cell alloreactivity, PBMC obtained from normal individuals were cocultured with irradiated allogeneic PBMC at a 2:1 cell ratio in the absence or presence of various concentrations of G-CSF (0, 10, 100 ng/ml). After 7 days of culture, viable cells were isolated from dead cells by Histopaque. These mononuclear cells (MNC: 1 × 105 cells) were then restimulated with irradiated allogeneic PBMC (1 × 105 cells) for 48 h in triplicate and then pulsed with 3H-thymidine for the last 18 h of culture. In some experiments, CD4+ cells isolated from MNC after primary MLR were used as effector cells of the secondary MLR. Cell proliferation was determined as described above.
To determine cytokine production, responder cells (1 × 106 cells) were cocultured with irradiated, allogeneic stimulator cells (1 × 106 cells) or with 10 μg/ml of Con A in 1 ml of complete medium in 24-well, flat-bottomed plates (Costar) at 37°C in 5% CO2 in duplicate. Cell-free supernatants were harvested after 48 h of culture and were stored at −80°C until use. ELISA kits (Human high sensitivity IL-4 ELISA kit, Human IFN-γ ELISA kit; R&D Systems, Minneapolis, MN, USA) were used to measure cytokine levels according to the manufacturer's protocols. Each supernatant was measured in duplicate by a microplate reader (Bio-Rad Labs, Hercules, CA, USA). Minimal detectable levels of IL-4 and IFN-γ were 0.090 pg/ml and 15.6 pg/ml, respectively.
Data represent mean ± standard error (s.e.). The statistical significance was analyzed by Student's paired t-test. Differences with a P value less than 0.05 were considered significant.
T cell proliferative responses to allogeneic stimulation and Con A were impaired in G-CSF-mobilized PBSC
We removed adherent cells from PBMC by plastic adherence, since the frequency of T cells in post-G-CSF PBMC was lower than that before G-CSF administration (41% vs 63%) due to contamination by a large number of monocytes (18% vs 38%), as has been reported.151617 Following plastic adherence, the frequency of T cells and monocytes was determined by FACS analysis in preG-NAC and G-NAC obtained from each individual. Depletion of adherent cells resulted in a decrease in the percentage of monocytes and an increase in the percentage of T cells. In preG-NAC, the percentages of CD3+, CD4+, CD8+ and CD14+ cells were 77 ± 3, 38 ± 4, 32 ± 5 and 9 ± 3%, respectively. Those in G-NAC were 73 ± 5, 39 ± 3, 28 ± 5 and 16 ± 6%, respectively. The frequency of each population was not significantly different between preG-NAC and G-NAC. The CD4 to CD8 ratio was also equivalent in the two populations. When cells were cultured with allogeneic stimulators for 7 days, T cell proliferation was reduced in G-NAC compared to that in preG-NAC (845 ± 342 c.p.m. vs 4119 ± 1814 c.p.m.), despite the equivalent cell compositions (Table 1). T cell proliferation in G-NAC was also significantly reduced compared to that in preG-NAC when percentages of control (preG-NAC) were used to summarize the differences (55 ± 21% of preG-NAC, P < 0.01). A similar hypoproliferative response was observed in G-NAC upon stimulation with Con A (654 ± 471 c.p.m. vs 1217 ± 468 c.p.m.) (58 ± 9% of control, P < 0.01).
T cell hypoproliferative response in G-NAC was associated with decreased production of both IFN-γ and IL-4
In order to investigate whether a reduced T cell alloresponse in G-NAC is associated with polarization of T cells towards a type 2 cytokine profile, as has been observed in murine studies,91011 IFN-γ and IL-4 levels in the culture supernatant were measured. The kinetics of IFN-γ and IL-4 production in G-NAC induced by either allogeneic stimulation or Con A stimulation were similar to those of preG-NAC. Our preliminary kinetic study of the production of IFN-γ and IL-4 demonstrated the level of these cytokines reached the maximum at 48 h of culture (data not shown). Production of cytokines in response to allogeneic stimulation was reduced in G-NAC compared to that in preG-NAC (IFN-γ: 157 ± 81 pg/ml vs 321 ± 51 pg/ml, IL-4: 0.62 ± 0.26 pg/ml vs 3.5 ± 2.2 pg/ml). These differences were statistically significant when percentages of control (preG-NAC) were used to summarize the differences (IFN-γ: 49 ± 21%, P < 0.05, IL-4: 55 ± 14%, P < 0.05) (Table 1). Con A-induced production of IFN-γ and IL-4 was also reduced in G-NAC.
Proliferative and cytokine responses were restored in CD3+ T cells positively selected from G-NAC
We then tested alloresponses of CD3+ cells positively selected from preG-NAC and G-NAC. Although positive selection using anti-CD3 mAb can stimulate T cells via the CD3/TCR complex, these cells did not proliferate or produce cytokines in a 7-day culture without further stimulation. Interestingly, proliferative and cytokine responses to allogeneic stimulation in purified CD3+ cells from G-NAC were equivalent to those from preG-NAC (Table 2).
T cell alloreactivity in MLR in the presence of post-G-CSF monocytes was less than that in the presence of pre-G-CSF monocytes
Based on the results of these experiments, we hypothesized that monocytes after G-CSF administration are responsible for the inhibition of T cell responses. We therefore performed add-back studies of monocytes to test suppressive function of post-G-CSF monocytes in MLR. Adherent cells were isolated from PBMC obtained before and after G-CSF administration. Viability of these purified monocytes always exceeded 95% as determined by trypan blue dye exclusion, and purity of monocytes always exceeded 90% as determined by CD14 positivity on flow cytometry. The frequency of CD14+ monocytes determined by FACS analysis was not different between pre-G-CSF monocytes and post-G-CSF monocytes. 1 × 105 of PBMC obtained prior to G-CSF administration were cultured with 1 × 105 irradiated third party PBMC for 7 days in the presence of autologous monocytes obtained before or after G-CSF administration. T cell proliferation in MLR was less when post-G-CSF monocytes were added to the cultures than when cultured in the presence of pre-G-CSF monocytes (Figure 1).
G-CSF did not affect MLR activity in vitro
Experiments were conducted to determine whether G-CSF directly modulates monocyte function in vitro. PBMC obtained from normal individuals were cultured for 7 days with irradiated third party allogeneic stimulators in the presence or absence of G-CSF. MNC recovered from primary culture or CD4+ cells isolated from these MNC were restimulated with alloantigen and T cell proliferation was measured 48 h later. As shown in Table 3, secondary MLR activity of MNC and CD4+ cells was not affected by the addition of G-CSF to the primary culture, suggesting that the mechanism of G-CSF-induced modulation of monocyte function is indirect.
Potential mechanisms for the lack of increase in the incidence and severity of acute GVHD in allogeneic PBSCT compared to BMT, despite the marked increase in T cell numbers in PBSC grafts, have been widely investigated. The majority of studies in humans has focused on reduced T cell alloreactivity in post-G-CSF PBMC in MLR.1314151617181920 However, the hyporesponsiveness of PBMC in MLR does not allow for precise conclusions about donor T cells, since the cell composition of post-G-CSF PBMC is notably different from steady-state PBMC due to contamination by a higher number of monocytes in post-G-CSF PBMC.151617 Therefore, adherent cells were roughly depleted from PBMC by plastic adherence in this study in an attempt to normalize T cell numbers. As a consequence of adherent cell depletion, frequencies of T cells and monocytes were similar in G-NAC and preG-NAC, although there were still about 10% CD14+ monocytes in NAC. Nonetheless, proliferative responses of G-NAC to alloantigen and Con A were significantly less than those of preG-NAC. This observation confirms previous studies demonstrating decreased responses of post-G-CSF PBMC to allogeneic stimulation and mitogens,1314151617181920 even when T cell and monocyte numbers were normalized.
Although murine models of PBSCT suggested that G-CSF treatment of the donor shifted the cytokine profile to favor a type 2 pattern in both donors and recipients after allogeneic PBSCT,91011 the cytokine profile of donor T cells following high-dose G-CSF administration has not been fully investigated in humans. Our studies demonstrated reduced production of a type 2 cytokine, IL-4, as well as a type 1 cytokine, IFN-γ, in G-NAC in association with a reduced T cell proliferative response. A previous study in humans demonstrated that levels of type 1 cytokines (IL-2 and IFN-γ) in the supernatant of an MLR were reduced when irradiated post-G-CSF PBMC were used as the stimulator.17 It suggests the reduced ability of post-G-CSF PBMC to stimulate allogeneic T cells.
Interestingly, T cell responses to allogeneic stimulation were completely restored in CD3+ T cells positively selected from G-NAC; proliferation and production of IFN-γ and IL-4 in CD3+ T cells were similar before and after G-CSF treatment. This confirms our preliminary study demonstrating normal proliferative responses of post-G-CSF CD4+ or CD8+ cells to those of pre-G-CSF corresponding cells,18 and a previous study demonstrating the restoration of T cell proliferative response to CD3 stimulation by the removal of CD14+ monocytes.20 It has also been shown that T cells in GM-CSF-mobilized PBSC have a depressed mitogenic response to PHA, but positively selected CD4+ and CD8+ T cells from PBSC showed normal mitogenic response.23 Collectively, these results suggest the lack of an intrinsic defect of T cells and the presence of suppressive effects of accessory cells in apheresis products, although we cannot rule out the possibility that positive selection procedures will restore T cell function.
We demonstrated that IFN-γ and IL-4 production was suppressed in G-NAC but normal in post-G-CSF CD3+ T cells. Therefore, polarization of donor T cells toward type 2 cytokine response by G-CSF treatment is not likely in humans as it is in mice. A couple of studies of the cytokine profile in humans using a reverse transcriptase-polymerase chain reaction (RT-PCR)-based method suggested that G-CSF-mobilized PBMC tend to constitutively express more type 2 cytokines in both donors and recipients of allogeneic PBSCT.2425 Recently, it was reported that G-CSF-mobilized PBSC obtained from cancer patients constitutively express significantly higher levels of IL-2, 4 and 10, IFN-γ and TNF-α as compared to normal PBMC.26 Furthermore, mRNA expression of the cytokines IL-2, 4 and 10 and IFN-γ in purified CD4+ and CD8+ cells from PBSC were higher compared with those from normal PBSC.27 Our data, however, do not agree with the results of these studies, probably due to the differences in the experimental methods used. Prior studies detected cytokine mRNA expression on unstimulated cells with a RT-PCR technique, while our study measured cytokines in the supernatant of stimulated cells by ELISA.
Our add-back studies of autologous monocytes in MLR confirm the role of post-G-CSF monocytes in the suppression of T cell proliferative responses in vitro as previously described,15162021 although we cannot deny the possibility that other cells, such as B cells and granulocytes in PBSC products, may also be suppressive. Suppressive effects of monocytes have been attributed to IL-10 production by a large number of monocytes,2027 as well as reduced expression of the costimulatory molecule CD86 on monocytes15 and impaired induction of the CD28 responsive complex on T cells in G-PBMC,21 which therefore provide suboptimal amounts of costimulatory signals. It was also shown that inhibition of monocytic IL-12 and TNF-α production by G-CSF indirectly attenuates IFN-γ release from T cells.28 A recent study suggests that primed Fas+ CD4+ T cells in G-CSF-mobilized PBSC interact with activated Fas ligand+ monocytes, resulting in apoptosis.29 The circulatory half-life of monocytes is approximately 3 days and monocytes can migrate into peripheral tissue where alloreactive responses take place.3031 Therefore, although there is no direct evidence that infused post-G-CSF monocytes affect T cells in vivo, it is possible that infused monocytes suppress alloreactivity of T cells in vivo. A comparative study of immune reconstitution after allogeneic BMT and PBSCT demonstrated that monocyte count early after PBSCT was greater than that after BMT, but it rapidly declined to the comparable level after BMT.6 It is tempting to speculate that the large numbers of G-CSF-mobilized monocytes contained in PBSC suppress alloresponses of T cells, leading to the relative decrease in acute GVHD, and that T cell alloresponses are restored as monocytes disappear, leading to the increase in incidence of chronic GVHD.32
We then asked whether G-CSF modulates monocyte function directly. We did not demonstrate a direct effect of G-CSF on PBMC in secondary MLR when G-CSF was added into a 7-day primary MLR culture. This confirms our previous study demonstrating no direct effect of G-CSF in primary MLR.18 We recently found that post-G-CSF monocytes were functionally and phenotypically different from steady-state monocytes (Sunami K et al, unpublished observation). These alterations, however, were not replicated by culturing monocytes with G-CSF in vitro. Thus, modulation of monocyte function seems to be induced by mobilization of functionally different monocytes or by complicated interactions of cells and/or humoral factors in vivo.
In summary, our results suggest that G-CSF treatment indirectly modulates monocyte function to inhibit T cell proliferative and cytokine responses to alloantigen and mitogen and that T cell functions can be restored by the depletion of these accessory cells.
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We are grateful to Drs Erik J Carson and Raymond J Hutchinson for helpful discussion and thank Kirin-Sankyo, Chugai Pharmaceuticals Co Ltd, and Kyowa Pharmaceuticals Co Ltd for kindly providing G-CSF. This study is supported in part by a grant-in-aid from the Ministry of Health and Welfare, the Ministry of Education, Science and Culture (06454348), and Uehara Memorial Foundation.
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Nawa, Y., Teshima, T., Sunami, K. et al. G-CSF reduces IFN-γ and IL-4 production by T cells after allogeneic stimulation by indirectly modulating monocyte function. Bone Marrow Transplant 25, 1035–1040 (2000) doi:10.1038/sj.bmt.1702402
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