| | Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Alloreactivity in Hematopoietic Cell Transplantation with Nonmyeloablative ConditioningReceived 21 June 2005; accepted 6 September 2005. Abstract Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)–2, IL-4, IL-10, IL-18, tumor necrosis factor α, and transforming growth factor β in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.
Introduction  Reduced-intensity or nonmyeloablative conditioning regimens in hematopoietic cell transplantation (HCT) have extended the use of this potentially curative treatment to patients who, because of age or comorbidities, were previously ineligible for HCT. Graft-versus-host disease (GVHD) remains one of the major complications associated with this treatment [1, 2], and further insight into the pathogenesis of GVHD is therefore important to develop approaches that can reduce the incidence of this complication. The release of inflammatory cytokines such as tumor necrosis factor (TNF)–α has been shown to be involved in the pathophysiology of GVHD [3, 4], and interleukin (IL)–10 and transforming growth factor (TGF)–β have been identified as important factors in the control of alloreactivity [5, 6, 7]. The role of cytokines in the development of GVHD in the clinical setting has therefore been explored in different ways. Gene polymorphisms that influence the production or effect of several cytokines have been related to the clinical outcome [8, 9, 10], and functional assays that measure cytokine production or cytokine gene expression in mixed lymphocyte cultures (MLCs) performed before transplantation have been used to predict clinical events such as acute GVHD [11, 12, 13]. After transplantation, the occurrence of GVHD has been compared with the cytokine levels in serum or plasma [14, 15, 16, 17, 18, 19, 20, 21, 22, 23] or with the cytokine gene expression in cells from peripheral blood [22, 24, 25, 26, 27, 28, 29, 30, 31]. The exact relationship among cytokines, alloreactivity, and the effect on the overall success of the treatment is, however, not completely elucidated, and only a few studies have included patients who have undergone transplantation with nonmyeloablative or reduced-intensity conditioning regimens [17, 24, 32, 33, 34, 35]. The results obtained in recipients of myeloablative conditioning may not be applicable in the nonmyeloablative setting because of the different patient populations treated and the requirement for a potent graft-versus-tumor (GVT) effect after nonmyeloablative conditioning. In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to analyze the gene expression of IL-2, IL-4, IL-10, IL-18, TNF-α, and TGF-β in peripheral blood mononuclear cells (MNCs) obtained from donors and recipients before and after HLA-identical HCT with nonmyeloablative conditioning. The results suggested that a high IL-10 messenger RNA (mRNA) level in MNCs obtained on day +14 and cultured in the presence of recipient or donor antigens was related to a low incidence of acute GVHD grades II to IV. The pretransplantation IL-10 mRNA level was higher in patients who experienced relapse than in those who did not. In conclusion, IL-10 may be related to reduced alloreactivity after HCT with nonmyeloablative conditioning. Although the reduced incidence of acute GVHD is beneficial for the patients, this may be at the expense of a reduced GVT effect. Materials and methods Patients and Donors Twenty-one patients with hematologic malignancies and their HLA-identical sibling donors were included. The patients, unique patient numbers (UPNs) 628, 633, 636, 645, 650, 656, 662, 669, 682, 688, 692, 694, 708, 715, 723, 733, 741, 760, 766, 780, and 796, underwent transplantation between 2000 and 2003 with peripheral blood stem cells after nonmyeloablative conditioning consisting of 2 Gy of total body irradiation with or without fludarabine 90 mg/m2 combined with cyclosporine and mycophenolate mofetil as posttransplantation immunosuppression [36, 37, 38]. The patients, donors, and volunteers gave written informed consent before inclusion, and the local ethics committee approved the study. Acute and chronic GVHD were diagnosed and graded according to standard criteria [39]. Patients who survived >80 days were evaluated for the occurrence of chronic GVHD. Preparation and Storage of MNCs MNCs were obtained 2 weeks before the start of conditioning from leukaphereses performed in all the patients and in 16 of the donors as recently described [40]. The MNCs obtained from the remaining donors, HLA-mismatched volunteers, and patients after transplantation were separated from heparinized peripheral blood samples by gradient centrifugation (Lymphoprep; Axis Shield PoC, Oslo, Norway). The MNCs were cryopreserved in liquid nitrogen in 50% medium (RPMI 1640 with 100 IU/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l-glutamine, all from Invitrogen, Tåstrup, Denmark), 40% heat-inactivated human serum from male AB donors (ICN Biomedicals, Aurora, OH), and 10% dimethyl sulfoxide. The patients were scheduled to have blood samples taken on days +7, +14, +21, +28, +42, +56, +90, +120, +180, +270, and +365. In Vitro Stimulation The MNCs were thawed, washed twice in medium, and resuspended in medium with 10% human serum (culture medium). Viable cells were counted by using the trypan blue exclusion method. To define the optimal incubation period, 2 experiments of 9 cultures containing 5 × 104 responder cells and 5 × 104 HLA-mismatched stimulator cells irradiated with 50 Gy from a cesium 137 source were performed in culture medium in round-bottomed 96-well microtiter plates. The cultures were incubated at 37°C in 7.5% carbon dioxide. Each day, except for day 7, the content of 1 well was removed and centrifuged, and the cell pellet was frozen. The gene expression of IL-18 was not examined in this experiment. The MNCs obtained before transplantation were cultured in duplicate in a final volume of 200 μL of culture medium in 96-well plates. The wells contained 5 × 104 unstimulated MNCs, 5 × 104 MNCs cultured in the presence of 3 μg/mL phytohemagglutinin (PHA), MLC with 5 × 104 responder MNCs and 5 × 104 irradiated HLA-identical MNCs (donor versus recipient and vice versa), or 5 × 104 responder MNCs and 5 × 104 irradiated HLA-mismatched third-party MNCs. The MNCs obtained after transplantation were unstimulated, stimulated with PHA, or cultured with irradiated donor, recipient, or third-party MNCs. The cultures containing irradiated HLA-mismatched cells or PHA were included as positive controls. All cultures were incubated for 72 hours, whereafter the plates were centrifuged at 800g for 3 minutes, and 2 × 75 μL of supernatant was stored at −20°C. The cell pellets were stored at −80°C. Responder Cell Composition The fractions of CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and monocytes present in the responder and stimulator cells were determined by 3-color flow cytometry on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) by using monoclonal antibodies obtained from Becton Dickinson. The percentages of donor chimerism of CD4+ and CD8+ T cells were determined as previously described [38]. IL-2 and IL-4 in Culture Supernatants The content of secreted bioactive IL-2 and IL-4 in each well was measured by use of the CTLL-2 cell line (American Tissue Culture Collection, Rockville, MD) and the CT.h4S cell line (a generous gift of W.E. Paul, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD), respectively, as described previously [41, 42]. The detection limits were 6 pg/mL for the CTLL-2 bioassay and 5 pg/mL for the CT.h4S bioassay. Generation of Complementary DNA Messenger RNA was extracted from the frozen cell pellets by using the Dynabeads mRNA DIRECT Kit (Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions. A negative sample containing culture medium only was included in each extraction. Complementary DNA (cDNA) was generated in GeneAmp tubes (Applied Biosystems, Foster City, CA) in a final volume of 20 μL by using 2 μL of 10× PCR Rxn Buffer (Invitrogen), 2 μL of 50 mmol/L MgCl2 (Invitrogen), 8 μL of deoxyribonucleoside triphosphate mix corresponding to a final concentration of 2.5 mmol/L (Ultrapure dNTP Set; Amersham Pharmacia Biotech, Piscataway, NJ), 1 μL of murine leukemia virus reverse transcriptase (50 U/μL; Applied Biosystems), 1 μL of ribonuclease inhibitor (20 U/μL; Applied Biosystems), 1 μL of random hexanucleotides corresponding to a final concentration of 0.25 μmol/L (DNA Technology, Århus, Denmark), and 5 μL of mRNA. Ten microliters of mineral oil (Applied Biosystems) was added to each tube, and cDNA was generated by using 30 minutes at 42°C followed by 5 minutes at 99°C. The cDNA was stored at −80°C. Cytokine Gene Expression Expression of β2-microglobulin, IL-2, IL-4, IL-10, IL-18, TNF-α, and TGF-β genes was determined by real-time quantitative multiplex RT-PCR by using predeveloped TaqMan assay reagents (control and target kits; Applied Biosystems) in a final volume of 25 μL in 96-well plates obtained from Applied Biosystems, by using the TaqMan ABI 7700 and 7900HT Sequence Detection Systems (Applied Biosystems). Each well contained 2.9 μL of cDNA, 7.1 μL of ribonuclease-free water, 12.5 μL of Master Mix (Applied Biosystems), 1.25 μL of primers and probes for β2-microglobulin (labeled with VIC® dye; Applied Biosystems), and 1 of the 6 cytokine genes (labeled with FAM™ dye; Applied Biosystems). After initial steps of 2 minutes at 50°C and 10 minutes at 95°C, 50 cycles of 15 seconds at 95°C followed by 1 minute at 60°C were performed. Sequence Detection System software version 2.0 was used for data analysis. The relative mRNA level of the cytokine was calculated as 2ΔCt, where ΔCt is the threshold cycle (Ct) value for β2-microglobulin minus the Ct value for the cytokine. A threshold was chosen for each plate so that the Ct values for both β2-microglobulin and for the cytokines were obtained in the log phase of the PCR reactions. This threshold was most often close to 0.2. ΔCt was rounded to the nearest whole figure. The lower limit of detection in this assay was arbitrarily set to a ΔCt of −15, which corresponds to a 3 × 10−5 times lower mRNA level of the cytokine than of β2-microglobulin. The reason for this was that ΔCt values lower than −15 were often observed when the Ct value for the cytokine was >40 cycles. Statistical Methods Except for the initial longitudinal experiment, only data representing the mean of duplicate cultures were used. The relative mRNA levels were compared by using the Friedman test for paired data and the Kruskal-Wallis test for unpaired data. In both cases, the Dunn correction for multiple comparisons was used for the post hoc tests. The Mann-Whitney test was used to compare the IL-10 mRNA level in cells from patients with or without acute GVHD or relapse. Nonparametric correlation (Spearman) was used to correlate continuous data. The data for clinical follow-up were last updated on March 17, 2005. Of the study cohort, 15 patients were followed up with blood samples for a full year, whereas the remaining 6 patients were followed up for a median of 120 days (range, 28-180 days). Samples from patients whose disease progressed or relapsed were not excluded from the study. A P value <.05 was considered significant. GraphPad Prism version 4 (GraphPad Software, San Diego, CA) was used for all calculations. Results Clinical Results The patient characteristics are listed in Table 1. Six patients (UPN 636, 656, 662, 688, 715, and 780) died a median of 160 days after transplantation (range, 72-659 days), and 6 patients (UPN 650, 682, 688, 708, 715, and 733) experienced progression or relapse at a median of 413 days (range, 56-728 days), as previously described [37]. The median follow-up in surviving patients was 1371 days (range, 762-1826 days). Two patients developed acute GVHD grade I, 10 patients developed grade II, 1 patient developed grade III, and 2 patients developed grade IV. Sixteen of the 20 evaluable patients developed limited or extensive chronic GVHD at a median of 216 days after transplantation (range, 100-476 days) [37]. Messenger RNA Levels and Protein Secretion of IL-2 and IL-4 We used 2 different real-time PCR platforms (the ABI 7700 and 7900HT) and found no differences in the results obtained (data not shown). On the basis of the initial longitudinal experiment, we concluded that 3 days (72 hours) of incubation would be optimal for the study of cytokine gene expression in the HLA-mismatched MLC (Figure 1). To make the experiments practical and comparable, all the subsequently performed cultures were incubated for 72 hours. The IL-2 and IL-4 mRNA levels and the results of IL-2 and IL-4 measurements in the supernatants were compared, and a high degree of correlation was observed for both cytokines (Spearman r = 0.78 and r = 0.84, respectively; P < .0001 in both cases; data not shown). The simultaneous detection of cytokine mRNA in the cell pellet and bioactive cytokine in the supernatant occurred in 36% of the cultures for IL-2 and in 23% for IL-4. In a large proportion of cultures (29% for IL-2 and 66% for IL-4), we were unable to detect either mRNA or cytokine in the supernatant, and only in 1% for IL-2 and 2% for IL-4 did we detect cytokine in the supernatant without detectable mRNA. In the remaining 34% of the cultures for IL-2 and 9% for IL-4, we detected mRNA without measurable cytokine in the supernatant. In these cultures, the median mRNA level was 16 and 11 times lower, respectively, than the level in the cultures in which IL-2 and IL-4 could be detected by the cell lines. We conclude that for IL-2 and IL-4, there is a relationship between the mRNA level and the amount of secreted protein. The measurement of IL-2 and IL-4 mRNA by real-time RT-PCR may be more sensitive than the detection of cytokine by the CTLL-2 and the CT.h4S cell lines. Pretransplantation Cytokine Gene Expression in MNCs from Patients and Donors The pretransplantation responses to the different stimuli were compared with the unstimulated cultures in the recipient and donor groups (Figure 2). For IL-2, the responses to HLA-identical MNCs were not different from the unstimulated cultures, but significant responses were observed to PHA in the recipients and to HLA-mismatched third-party MNCs and PHA in the donors (Figure 2a). For IL-4 and TNF-α, the responses to PHA could be distinguished from the unstimulated cultures (Figure 2b and c), whereas for IL-10 and TGF-β, this was observed only in the donor MNCs (Figure 2d and e). The mRNA level of IL-18 was significantly lower in the PHA-stimulated cultures than in the unstimulated cultures (Figure 2f). These findings indicate that for the cytokines examined in this study, the analysis of cytokine mRNA in an HLA-identical MLC performed with cells obtained before transplantation from patients and donors does not yield additional information compared with the analysis of the unstimulated cells. That this is not due to a general unresponsiveness is illustrated by the responses to third-party cells and to PHA. When comparing results in patients and donors, we observed higher mRNA levels of IL-10 and TGF-β in the unstimulated cultures containing recipient cells (Figure 2d and e), and in the cultures stimulated with HLA-mismatched MNCs, the IL-2 mRNA level was lower and the mRNA levels of IL-10, TGF-β, and IL-18 were higher when the responder cells were of recipient origin than when donor cells were used (Figure 2a and d-f). The differences between patients and donors in the cytokine mRNA levels of the unstimulated cells and in the ability to respond to third-party antigens could represent functional differences of the responding cells or differences in the composition of the responder MNCs. We examined the composition of the responder MNCs and found a lower percentage of CD4+ T cells in the recipient MNCs (median, 18%; range, 0.3%-33%) than in the donor MNCs (median, 44%; range, 27%-58%; P < .001; Kruskal-Wallis test with the Dunn correction for multiple comparisons). No differences could be demonstrated for the other cell types investigated (data not shown). To analyze whether the CD4+ T-cell number influenced the results shown in Figure 2, we examined the mRNA level of IL-2 per CD4+ T cell, because this cytokine is primarily produced by activated CD4+ T cells. In the wells containing third-party stimulator cells, we found no difference in the level of IL-2 per CD4+ T cell when responder cells of recipient and donor origin were compared (P = .25; Mann-Whitney test), a finding that was in contrast to the observation in Figure 2a. In conclusion, the cell composition is a factor that is likely to confound the results of cytokine mRNA analysis of unfractionated MNCs. Cytokine Gene Expression after Transplantation After transplantation, stimulation with PHA increased the mRNA levels of IL-2, IL-4, IL-10, and TNF-α and decreased the IL-18 mRNA level (data not shown). Stimulation with HLA-mismatched MNCs increased the IL-2 mRNA level and slightly decreased the IL-10 mRNA level (data not shown). The cytokine mRNA levels in the cultures stimulated with recipient or donor MNCs resembled the unstimulated cultures (TNF-α, TGF-β, and IL-4; Figure 3) or showed minor differences (IL-18 and IL-2; Figure 3a). It is interesting to note that the IL-10 mRNA level early after transplantation was higher in the cultures stimulated with recipient or donor MNCs when compared with the unstimulated cultures (P = .0006 and P = .006, respectively, on day +14; Wilcoxon signed rank test), thus indicating that in contrast to the pretransplantation experiments, we were able to detect significant responses to irradiated HLA-identical MNCs after transplantation (Figure 3b). We have previously found that patients with a high donor CD8+ T-cell count on day +14 had a high risk of development of acute GVHD grades II to IV [38]. The mRNA levels of the immunoregulatory cytokines IL-10 and of TGF-β were therefore further examined with relation to the development of acute GVHD (Figure 4). Whereas the mRNA level of TGF-β in patients who developed acute GVHD grades 0 or I and grades II to IV did not depend on the stimulus used, this again was not the case for IL-10 in the early period after transplantation (Figure 4). The data for day +14 were analyzed, and there was no difference in the IL-10 mRNA levels between the patients who developed acute GVHD grades II to IV and patients who did not develop clinically significant acute GVHD in the unstimulated cultures or in the cultures stimulated with HLA-mismatched MNCs (P = .49 and P = .64, respectively; Mann-Whitney test; Figure 4a and b). In contrast, in the cultures stimulated with recipient or donor MNCs, the IL-10 mRNA levels were lower on day +14 in the cultures from patients who subsequently developed acute GVHD grades II to IV when compared with cultures from the patients with acute GVHD grades 0 or I (P = .005 and P = .004, respectively; Mann-Whitney test; Figure 4c and d). Not only mRNA levels, but also the composition of the responder cells, varied during the first year after transplantation. Both the monocyte (CD14+) fraction and the fraction of CD8+ T cells began to deviate between the 2 groups of patients after the onset of acute GVHD, which in most patients occurred between day +28 and +42 (Figure 4e and f ). The responder cell composition was able to influence the results as observed in the pretransplantation setting. We therefore find that the responder cell composition cannot be excluded as a reason for the differences between patients with and without acute GVHD that can be observed from day +28 onward. The differences appear for TGF-β regardless of the stimulus used and for IL-10 in the unstimulated cultures and in the wells containing HLA-mismatched MLC (Figure 4a-d). The finding that peripheral blood MNCs from patients who subsequently developed acute GVHD grades II to IV showed lower levels of IL-10 mRNA on day +14 both in response to irradiated recipient and donor cells was surprising, because we would have expected a more specific response. We were therefore interested in determining whether the IL-10 mRNA originated from the stimulator cells or from responder cells of donor or recipient origin. The patients were not complete hematopoietic chimeras at this time point, having a median of 71% donor CD4+ T cells (range, 40%-97%) and a median of 64% donor CD8+ T cells (range, 19%-98%) in the peripheral blood. The results in the cultures performed before transplantation with unstimulated cells of recipient or donor origin were correlated with the results on day +14 (Figure 5). Whereas a high degree of correlation was observed between the recipient cells obtained before transplantation and the cultures stimulated with irradiated recipient or donor cells on day +14 (Figure 5a and b), no correlation was observed between the IL-10 mRNA level of the donor cells before transplantation and the level in the cultures performed on day +14 (Figure 5c and d). In conclusion, we find that the IL-10 mRNA detected on day +14 is more likely to originate from the recipient responder cells than from the donor responder cells. The observation that the correlation with the pretransplantation recipient values is present both in cultures stimulated with recipient and donor MNCs on day +14 makes it unlikely that the IL-10 mRNA determined on day +14 originates from the irradiated stimulator cells. To further study the relationship between acute GVHD and cells of recipient and donor origin, we used the values for CD8+ and CD4+ T-cell chimerism in each patient to calculate the numbers of responder recipient and donor T cells in the cultures performed on day +14. The donor T-cell number did not differ between patients with and without acute GVHD (grades 0-I: median, 3300 CD8+ T cells and 11400 CD4+ T cells; grades II-IV: median, 3400 CD8+ T cells and 9100 CD4+ T cells). In contrast, there were significantly more recipient CD8+ T cells in the cultures with cells from patients who did not develop acute GVHD (median, 3600 cells; range, 600-6000 cells) when compared with the cultures from patients who developed acute GVHD grades II to IV (median, 1400 cells; range, 100-2400 cells; P = .016; Mann-Whitney test). The median recipient CD4+ T-cell number was also higher in cultures with cells from patients without development of acute GVHD (median, 6100 cells; range, 1100-10600 cells) when compared with the number in cultures with cells from patients who developed acute GVHD grades II to IV (median, 2000 cells; range, 200-10500 cells), but this difference was not significant (P = .21; Mann-Whitney test). On day +14, data were available for 16 cultures performed with recipient stimulator cells and for 12 cultures performed with donor stimulator cells (Figure 5). Pretransplantation data were available in all patients, and when we examined the relationship between the occurrence of acute GVHD and the pretransplantation level of IL-10, we found a trend toward higher levels of IL-10 mRNA in the unstimulated recipient MNCs from patients who did not develop clinical significant acute GVHD when compared to the levels in patients who developed acute GVHD grades II to IV (Figure 6a). The IL-10 mRNA levels of the unstimulated donor MNCs obtained before transplantation showed no difference according to the subsequent occurrence of acute GVHD (Figure 6a). In addition, the patients whose disease progressed or relapsed had a higher pretransplantation level of IL-10 mRNA in their unstimulated MNCs than the patients whose disease did not progress (Figure 6b). Again, no difference was observed in the donor cells (Figure 6b). Because of the high incidence of chronic GVHD in this cohort and the long time intervals between the acquirement of blood samples after 6 months, we did not attempt to analyze the relationship between chronic GVHD and cytokine gene expression. Discussion In this study, we have investigated whether real-time RT-PCR–based quantification of cytokine gene expression in MNCs obtained from recipients and donors before transplantation and from the recipients sequentially after transplantation can contribute to a better understanding of the immune responses underlying GVHD and the GVT effect in patients with hematologic diseases who undergo transplantation with a nonmyeloablative conditioning regimen. A high degree of correlation between the level of mRNA and the amount of bioactive cytokine detected in the culture supernatants was observed for IL-2 and IL-4. However, the secretion of IL-10 is regulated by posttranscriptional mechanisms [43], and the biological activity of TGF-β is also subject to regulation [44], thus indicating that the amount of bioactive cytokine may not be directly estimated from the steady-state mRNA level. Before transplantation the patients had a lower fraction of CD4+ T cells in their MNCs than the donors. This factor was likely to influence the results because although the level of IL-2 mRNA in an HLA-mismatched MLC was higher when the responder cells were of donor origin than when recipient cells were used, the level of IL-2 mRNA per CD4+ T cell was not different. We observed a higher level of IL-10 mRNA in the unstimulated recipient MNCs when compared with the unstimulated donor MNCs. This may also be due to differences in cell composition, because IL-10 is produced by a variety of cells of the hematopoietic system, including monocytes, T cells, and B cells [45]. In addition, increased serum levels of IL-10 and increased IL-10 gene expression have been detected in patients with hematologic malignancies [46], but whether the IL-10 is produced by the malignant cells or by other cell populations remains to be established [45]. In the cultures performed after transplantation, we also observed that the cytokine gene expression was potentially affected by the responder cell composition. The cell composition of peripheral blood MNCs changes dramatically during reconstitution after HCT. In other studies of cytokine gene expression in MNCs or buffy coats after HCT, the cell composition was not determined [22, 24, 25, 26, 27, 28, 29, 30, 31]. In one study of in vitro cytokine production, no differences in cell composition were observed between patients with and without chronic GVHD, but in this study all the patients were examined >180 days after transplantation, when the cell composition may be more stable [47]. One way to overcome the effect of the composition of the MNCs could be to study isolated cell populations after HCT, as has been done by other investigators [34, 35, 48, 49]. We have used different stimuli and have observed that for IL-10, the stimulation with HLA-identical MNCs changes the pattern of gene expression when compared with the unstimulated cells. We observed higher IL-10 mRNA levels in cultures stimulated with irradiated recipient or donor cells on day +14 in patients who did not develop clinically significant acute GVHD when compared with cultures from patients who subsequently developed acute GVHD grades II to IV. In addition to this, we found it more likely that the IL-10 mRNA detected on day +14 originated from the recipient fraction of the responder cells than from the donor fraction. It is not clear why the recipient cells should express IL-10 in response to irradiated recipient stimulator cells on day +14, but one explanation could be that the irradiated recipient cells induce an autoreactive response [50]. We have not included an autologous control in the pretransplantation experiments, ie, recipient MNCs stimulated with irradiated recipient MNCs and the same for the donor. However, the recipient cells express IL-10 in response to all the stimuli used before transplantation (Figure 2d), and in our opinion it would therefore be unlikely that the mRNA levels of IL-10 should be low in an autologous MLC with recipient cells, but this issue needs further clarification. We also observed more recipient CD8+ T cells in the cultures performed with cells obtained on day +14 from patients who did not develop acute GVHD when compared with the cultures with cells from patients who developed acute GVHD grades II to IV. If the IL-10 mRNA detected in this study originated from the recipient cells, the observed difference in IL-10 mRNA levels of patients with and without acute GVHD could in theory be due to differences in the level of chimerism of T cells or other cell types. However, another hypothesis could be that the cytokine milieu early after transplantation could influence the rate of development of donor hematopoietic chimerism by affecting the alloreactive potential of the donor cells. The studies of IL-10 levels in serum or IL-10 gene expression after transplantation have come to different results regarding the relationship between IL-10 and acute GVHD. High levels of IL-10 have been associated with tolerance [49] and a lower incidence of acute GVHD [22, 29, 31]. However, increased IL-10 levels have also been observed before or during acute GVHD [14, 18, 19, 20, 21, 26, 51]. Because a major biological function of IL-10 is to oppose inflammatory responses [45], both the timing of samples and the level of inflammation induced by the conditioning regimen are likely to influence the results obtained. With the conditioning regimen used in this study, acute GVHD develops rather late after transplantation [36, 37], and most patients experience only minor complications during the first 4 weeks after transplantation. In this setting, we hypothesize that a high level of IL-10 in the recipients may limit the alloreactive potential of the donor cells, thus leading to a low incidence of acute GVHD. We observed high levels of IL-10 mRNA in the pretransplantation samples of patients who experienced relapse or progression and a trend toward lower levels of IL-10 mRNA in the samples of the patients who developed acute GVHD grades II to IV. High spontaneous IL-10 production by MNCs before transplantation has previously been associated with fewer transplant-related complications, including acute GVHD [52, 53]. Studies of gene polymorphisms in the IL-10 promoter region have also suggested that recipients who are high IL-10 producers are protected against severe acute GVHD [54, 55, 56, 57, 58] and death in remission [58]. To our knowledge, our data are the first to indicate that a high IL-10 gene expression by recipient cells before transplantation is associated with relapse or progression after HCT. Most of the previous studies were performed in recipients of myeloablative conditioning in patients who were likely to be in remission at the time of transplantation. In HCT with nonmyeloablative conditioning, the curative effect relies on the infused donor cells, and only a minority of the patients are in complete remission at the time of transplantation. Factors that tend to reduce the alloreactive potential of the donor cells may therefore have negative effects on the control of the malignant disease in these patients. Because of the high number of comparisons performed in this study, we cannot completely exclude that the results obtained for IL-10 are accidental findings. However, because alloreactivity is a central element of HCT with nonmyeloablative conditioning, we find that our study can contribute to the generation of hypotheses concerning the regulation of alloreactivity in this setting. These hypotheses may then be tested in larger cohorts of patients. In conclusion, we find that detection of cytokine gene expression in MNCs by real-time quantitative RT-PCR is an interesting tool in the study of immune responses after HCT. Several factors are likely to influence the results obtained, and in this study we have identified the composition of the MNCs as such a factor. Our results suggest a role for IL-10 as an inhibitor of alloreactivity after nonmyeloablative HCT. This inhibition may have dual effects by limiting the degree of acute GVHD and, at the same time, increasing the probability of relapse. Acknowledgments We thank Anne Bjørlig for excellent technical assistance with sample preparation and determination of cytokine gene expression. This work was supported by the Danish Cancer Society, the Gangsted Foundation, Fabrikant Vilhelm Pedersen og Hustrus Mindelegat, the Danish Medical Research Council, and the Novo Nordisk Foundation. References 1.
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1 The Lymphocyte Research Laboratory, Department of Hematology, Rigshospitalet, Copenhagen, Denmark 2 The Tissue Typing Laboratory, Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark 3 The Blood Bank, Department of Clinical Immunology, Rigshospitalet, Copenhagen, Denmark  Correspondence and reprint requests: Søren L. Petersen, MD, The Lymphocyte Research Laboratory, Department of Hematology L 4041, Rigshospitalet, Blegdamsvej 9, DK-2100, Copenhagen, Denmark.
PII: S1083-8791(05)00628-2 doi:10.1016/j.bbmt.2005.09.002 © 2006 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved. | |
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