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Clinical Cancer Research 13, 2916-2922, May 15, 2007. doi: 10.1158/1078-0432.CCR-06-2602
© 2007 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

The Graft Content of Donor T Cells Expressing {gamma}{delta}TCR+ and CD4+foxp3+ Predicts the Risk of Acute Graft versus Host Disease after Transplantation of Allogeneic Peripheral Blood Stem Cells from Unrelated Donors

Caroline Pabst, Holger Schirutschke, Gerhard Ehninger, Martin Bornhäuser and Uwe Platzbecker

Authors' Affiliation: Medizinische Klinik und Poliklinik I, Universitätsklinikum "Carl Gustav Carus" Dresden, Dresden, Germany

Requests for reprints: Uwe Platzbecker, Medizinische Klinik und Poliklinik I, Universitätsklinikum "Carl Gustav Carus" Dresden, 01307 Dresden, Germany. Phone: 49-351-458-4190; Fax: 49-351-458-5362; E-mail: Uwe.Platzbecker{at}uniklinikum-dresden.de.


   Abstract
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Materials and Methods
 Results
 Discussion
 References
 
Purpose: Recently, high numbers of regulatory T cells within the stem cell graft were described to be associated with less graft-versus-host disease (GVHD) after related peripheral blood stem cell transplantation (PBSCT). Studies in mice also suggest a distinct role of {gamma}{delta}TCR+ T cells in mediating GVHD. Therefore, the aim of this study was to define the yet-unknown role of regulatory and {gamma}{delta}TCR+ T cells in human PBSCT from unrelated donors.

Experimental Design: The frequency of both T-cell subsets within the graft was analyzed in 63 patients receiving unrelated allogeneic PBSCT. The respective amounts were quantified by flow cytometry and PCR and further correlated with clinical outcome.

Results: The grafts contained a median of 11.2 x 106/kg CD4+foxp3+ and 9.8 x 106/kg {gamma}{delta}TCR+ T cells, respectively. Patients receiving more CD4+foxp3+ cells had a lower cumulative incidence of acute GVHD II-IV (44% versus 65%, P = 0.03). Interestingly, in patients who received higher concentrations of donor {gamma}{delta}TCR+ T cells, acute GVHD II-IV was more frequent (66% versus 40%, P = 0.02). In multivariate analysis, only the graft concentration of {gamma}{delta}TCR+ T cells (P = 0.002) and a positive cytomegalovirus status of the recipient (P = 0.03) were significantly associated with the occurrence of acute GVHD II-IV.

Conclusion: Graft composition of T-cell subsets seems to affect the outcome of patients receiving allogeneic PBSCT from unrelated donors. Therefore, selective manipulation or add-back of particular subsets might be a promising strategy to reduce the incidence of GVHD.

Although allogeneic hematopoietic stem cell transplantation (HSCT) can provide long-term disease control in many patients with hematologic malignancies, its use is often complicated by severe graft-versus-host disease (GVHD). Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC) from allogeneic donors have become the most widely used source of hematopoietic cells for transplantation therapy (1). Unfortunately, it is associated with a higher frequency of chronic GVHD compared with bone marrow stem cell grafts (2). The reason for this increase is not entirely clear. Although previous clinical studies have investigated several donor graft characteristics influencing the incidence of GVHD (3–5), including CD34+ cell dose, the results are disparate (6, 7) and also seem to depend on the type of conditioning therapy (8). Recently, a novel T-cell subset named "regulatory T cells" (Treg) has been defined on the basis of CD4 surface and cytoplasmic foxp3 expression. Tregs inhibit autoreactive T-cell proliferation and suppress B-cell responses (9), thereby mediating graft tolerance while preserving a graft-versus-malignancy effect in the clinical setting of allogeneic HSCT (10). Although there are some limited and conflicting reports on the role of Tregs after related PBSC transplantation (PBSCT) (11, 12) their role after allogeneic transplantation from unrelated donors in man is unknown. Given the greater frequency of acute and chronic GVHD and treatment-related mortality (TRM) after transplants from alternative donors, there is a significant clinical need for better understanding of how Tregs interact within the host immune system. Other recent results suggest that the small subset of T cells expressing {gamma}{delta}T-cell receptor (TCR) participate in GVHD. The majority of data were obtained in mice and suggest that {gamma}{delta}TCR+ T cells promote GVHD induction (13–16), although the results vary in their implications (17, 18).

We therefore investigated the impact of Tregs and also of {gamma}{delta}TCR+ T cells in PBSC grafts on the incidence of GVHD, TRM, relapse, and overall survival after unrelated matched PBSCT in 63 patients with hematologic malignancies.


   Materials and Methods
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 Materials and Methods
Results
 Discussion
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Patients and donors. All patients and their respective unrelated donors received their transplants between November 1999 and July 2005 at the University Hospital of Dresden. Written informed consent for the respective research was obtained from each patient and donor, and the institutional review board of the Dresden University Hospital reviewed and approved the study. Patient characteristics are described in Table 1 .


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  		Table 1. Patient and graft characteristics of 63 patients receiving an unrelated PBSC graft

 
A total of 63 PBSC grafts mobilized from unrelated volunteer donors by G-CSF treatment were analyzed for the frequency of Tregs and {gamma}{delta}TCR+ T cells to study their impact on the outcome of the respective recipient after PBSCT. Aliquots of the grafts had been cryopreserved at transplant by controlled-rate freezing in autologous plasma containing 10% DMSO and stored in liquid nitrogen before analysis.

HLA typing. DNA-based HLA typing of donor and recipient was done using high resolution (four digits) for HLA-A, B, DRB1, and DQB1 and intermediate resolution (two digits) for HLA-C. As a consequence, patient and donor pairs were matched at least in 9 out of 10 alleles (n = 17; 27%), whereas a complete match (10 out of 10) existed in 46 (73%) patient-donor pairs.

Transplantation protocol. The intensive conditioning protocols applied before PBSCT were total body irradiation (TBI) with a total dose of 12 Gy plus cyclophosphamide (60-120 mg/kg, n = 23), TBI (8 Gy) plus fludarabine (90-120 mg/m2, n = 17) or busulfan (total dose 16 mg/kg) plus fludarabine (120-150 mg/m2, n = 23).

No ATG was used during conditioning, and all patients received unmanipulated PBSC on day 0 containing a median of 7.6 x 106/kg CD34+ cells (range, 3.4-17.9). All patients received cyclosporine A (CsA) and methothrexate (15 mg/m2, day 1; 10 mg/m2, days 3, 6, and 11 [n = 61]; or 10 mg/m2, days 1, 3, and 6 [n = 2]) as standard prophylaxis for GVHD. In the absence of GVHD, CsA was tapered from day 100 onward.

Regimen-related toxicity was scored using the Common Toxicity Criteria (CTC) version 3.0 of the National Cancer Institute.1 Acute and chronic GVHD were diagnosed and graded using established criteria (19, 20).

Flow cytometry. Cryopreserved aliquots of the donor grafts were thawed in a 37°C water bath, washed, and resuspended in cold medium containing PBS buffer and 5% heat-inactivated FCS (Invitrogen). Viability was verified with Trypan blue and reached on average >85% of all cells.

Cell surface antigens were stained using the following series of antihuman antibodies: CD4-PE, CD3-PE, and CD25-PE-Cy5 (Becton Dickinson), {gamma}{delta}TCR+-FITC (Becton Dickinson), CD8-FITC (Beckman Coulter), and CD4-FITC (DakoCytomation). The following antibodies were used as isotype controls: immunoglobulin G1 (IgG1)–PECy5 (Becton Dickinson), IgG1-FITC, IgG1-PE (Beckman Coulter), and IgG2a-PE (Hölzel Diagnostica).

The frequency and phenotype of the different T-cell subsets were analyzed using a three-color FACSCalibur Cytometer using CellQuest Pro software (Becton Dickinson). To quantify Tregs, intracellular expression of foxp3 was analyzed using a FITC-conjugated antibody to human foxp3, clone PCH101, and a FITC-conjugated rat antibody to human IgG2a as isotype control (eBioscience). Permeabilization and staining were done according to the manufacturer's protocol. Tregs were defined as CD4+foxp3+ given that foxp3 expression correlates well with the regulatory activity in human CD4+ cells (21). After gating on the lymphocyte population, we considered CD4+ and CD3+ cells separately. First, we gated on all CD4+ cells and determined the percentage of foxp3+ as well as CD25+ cells within the CD4+ population. In fact, we could not reliably differentiate between a high and medium/low CD25 expression on CD4+ T cells in all grafts. Therefore, the total amount of CD4+CD25+ T cells was considered. Furthermore, by gating on CD3+ lymphocytes, the percentage of {gamma}{delta}TCR+ cells out of CD3+ cells was determined. The absolute numbers of T cell subsets in the grafts were calculated based on the known absolute lymphocyte count of the stem cell product.

RNA isolation and cDNA synthesis. Total cellular RNA was isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. About 1 µg RNA was reverse transcribed for cDNA synthesis using the SuperScript II Reverse Transcriptase (Invitrogen), and cDNA was stored at –20°C until use.

Quantitative foxp3 PCR. For quantitative PCR of the foxp3 gene, equal amounts of cDNA for each sample were added to a prepared master mix containing TaqMan Universal PCR Master Mix (Applied Biosystems) and probes for foxp3 (Hs00203958; Applied Biosystems). All samples were analyzed at least in duplicate and are reported as mean values. ABL was used as housekeeping reference gene using the following primers: 5'-GGTTTGGGCTTCACACCATTC-3' (forward), 5'-CCCAACCTTTTCGTTGCA C-3' (reverse), 5'-(FAM)-AGCCTAAGACCCGGAGCTTTTCACC-(TAMRA)-3'(probe). The amounts of both transcripts were determined by comparison with calibration curves obtained by serial dilution of plasmids containing the full-length cDNA of each gene. The respective amount of foxp3 of each sample was divided by the endogenous reference (ABL) amount to obtain a normalized target value. All real-time quantitative PCR reactions were done on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) that captured the fluorescent signal and generated a real-time amplification plot. Thermal cycling conditions comprised an initial UNG incubation at 50°C for 2 min, AmpliTaq Gold activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min.

Statistical analysis. Initially, we considered each cell-type variable independently in a univariate analysis using cumulative incidence estimates (22) for the incidence of acute and chronic GVHD, TRM, and relapse and the method of Kaplan and Meier for estimates of overall survival (23). To estimate the correlation between cell types, Spearman's correlation coefficient was used. Comparison of the two groups, acute GVHD 0-I and II-IV, was done with Man Whitney U test. Gaussian distribution was tested with the Kolmogorov-Smirnov test. {chi}2 and Fisher test were used to estimate the significance of the cumulative effect of Tregs and {gamma}{delta}TCR cells. All P values are two tailed, and P values below 0.05 were considered to be significant. Non–cell-type variables were defined as patient age, cytomegalovirus (CMV) status, female donor/male recipient, and the degree of HLA compatibility. In a following multivariate analysis, cell-type variables, which had been shown to be associated with outcome, were analyzed again after controlling for non–cell-type variables.


   Results
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 Materials and Methods
 Results
Discussion
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We investigated the relationship between CD3+, CD4+foxp3+ (Tregs), CD4+CD25+, and {gamma}{delta}TCR+ T lymphocytes, as well as total nucleated and CD34+ cell dose in 63 PBSC grafts with regard to clinical outcome after allogeneic unrelated PBSCT, including the incidence of acute and chronic GVHD, TRM, relapse, and overall survival.

Graft characteristics. The general cellular composition including CD34+ cell dose of 63 PBSC grafts is given in Table 1. After cell surface and intracellular staining, a median of 6.1% (range, 2.2-16.7) of the CD4+ T cells were stained positive for foxp3. As a result, the absolute number of infused Tregs (CD4+foxp3+) was 2.8-33.5 x 106/kg (median, 11.3). We next determined foxp3 and ABL mRNA expression in 53 samples by quantitative PCR and compared the ratio foxp3/ABL with the absolute number of Tregs in the graft as determined by flow cytometry. Indeed, a significant correlation between foxp3 gene and protein expression (r = 0.54; P = 0.007, data not shown) was obtained, which supports the flow cytometry approach of quantifying Tregs. Additonally, 2.8% (range, 0.9-10.8) of all CD3+ T cells coexpressed the {gamma}{delta}TCR, which was not different from fresh PBSC grafts (2.6%; range, 0.8-21; n = 8) and peripheral blood of healthy donors (2.7%; range, 2.5-2.7; n = 3). Finally, the frequency of foxp3+, {gamma}{delta}TCR+, and CD34+ cells in the grafts showed a Gaussian distribution (data not shown).

Acute GVHD. Among 63 patients, 29 (46%) developed acute GVHD (aGVHD) grade 0-I, whereas 34 (54%) patients developed grade II-IV. Figure 1 provides a representative comparison between two patients who either developed GVHD grade I or III, respectively, with regards to the T-cellular composition of the graft received.


Figure 1
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  		Fig. 1. Phenotypic characterization of Treg (A, D, and G), CD4+CD25+ (B, E, and H) and {gamma}{delta}TCR+ T cells (C, F, and I) in two PBSC grafts. Shown are the characteristics of two different PBSC grafts. Phenotypic characterization of the grafts was done after gating on the total lymphocyte population. One patient received a graft (A-F) and developed acute GVHD grade I. D-F, results for Tregs, CD4+CD25+, and {gamma}{delta}TCR+ T cells, respectively, after gating on CD4+ (A and B) or CD3+ (C) cells. G-I, phenotypic characterization of another graft (patient experiencing aGVHD III) again after gating on CD4+ and CD3+ cells (dot plots not shown). The upper right quadrants provide the percentage of Treg, CD4+CD25+, and CD3+{gamma}{delta}TCR+ cells out of all lymphocytes (A-C) or within CD4+ (D, E, G, and H) and CD3+ (F and I) cells, respectively.

 
In univariate statistical analysis, Treg dose below the median was significantly associated with an increased cumulative risk of developing aGVHD II-IV (cumulative incidence 65% versus 44%, P = 0.03; Fig. 2 ). In line with this observation, the amount of Tregs in the grafts of patients who subsequently developed aGVHD grade 0-I was significantly higher than in grafts of patients who developed aGVHD grade II-IV (Fig. 3 ; median, 15.2 versus 8.7 x 106/kg; P = 0.01), whereas the absolute number of CD4+ T cells in the two groups did not show statistical differences (data not shown). Similarly, lower counts of CD4+CD25+ cells significantly correlated with an increased risk of aGVHD II-IV [5.3 (1.9-18.6) versus 6.8 (1.8-18.5) x 106/kg; P = 0.02; cumulative incidence, 64% versus 43%; P = 0.03]. A trend in the same direction was observed for the CD4+CD25+foxp3+ population [3.3 (0.3-12.6) versus 4.1 (1.3-13); P = 0.2; cumulative incidence, 60% versus 46%; P = 0.2].


Figure 2
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  		Fig. 2. Cumulative incidence estimates of acute GVHD grade II-IV according to donor Treg cell dose in the graft. Patients receiving Treg (CD4+foxp3+) cell counts below median (<11.25 x 106/kg) developed more aGVHD II-IV than patients receiving counts above median (cumulative incidence 65% versus 44%, P = 0.03).

 

Figure 3
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  		Fig. 3. Impact of transplanted Treg cell dose on acute GVHD. Shown is the Treg (CD4+foxp3+) cell dose in the grafts of patients developing aGVHD grade 0-I (left) and patients developing aGVHD grade II-IV (right).

 
Whereas the overall CD3+ cell dose was not correlated with the extent of aGVHD II-IV (data not shown), counts of {gamma}{delta}TCR-expressing CD3+ cells above the median showed a significant correlation with an increased cumulative risk of aGVHD II-IV (66% versus 40%; P = 0.02; Fig. 4 ). We then compared the absolute number of CD3+ and CD3+{gamma}{delta}TCR+ T cells in the grafts of patients who developed aGVHD grade 0-I and of patients who developed aGVHD grade II-IV. Similarly, there was no statistical difference in the number of CD3+ T cells (median, 3.3 versus 3.3 x 108/kg; P = 0.8), yet the count of CD3+{gamma}{delta}TCR+ T cells in the grafts of patients who developed GVHD grade II-IV was significantly higher than of patients who developed GVHD grade 0-I (Fig. 5 ; median, 12.4 x 106/kg; range, 3.5-36.2 versus median, 8.2 x 106/kg; range, 1.4-20.8; P = 0.03). There was no significant correlation between Tregs and {gamma}{delta}TCR+ T cells in the graft (r = 0.17; P = 0.16, data not shown).


Figure 4
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  		Fig. 4. Cumulative incidence estimates of acute GVHD grade II-IV according to donor {gamma}{delta}TCR+ T-cell cell dose in the graft. Patients receiving {gamma}{delta}TCR+ cell counts above median (>9.79 x 106/kg) developed more aGVHD II-IV than patients receiving counts below median (66% versus 40%, P = 0.02).

 

Figure 5
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  		Fig. 5. Impact of the transplanted {gamma}{delta}TCR+ T-cell cell dose on acute GVHD. {gamma}{delta}TCR+ T-cell dose in the graft of patients developing aGVHD grade 0-I (left) and of patients developing aGVHD grade II-IV (right).

 
The total nucleated cell dose infused had no impact on aGVHD (data not shown), whereas a lower CD34+ cell dose was significantly associated with an increased cumulative risk of aGVHD II-IV (63% versus 38%, P = 0.04). Concurrently, patients developing grade 0-I aGVHD had received significantly more CD34+ cells per kilogram [median, 8.7 (3.5-16.0)] than patients with more severe aGVHD [6.7 (4.0-17.9); P = 0.03]. Interestingly, there was a positive correlation between CD34+ and Treg cell dose (r = 0.34; P = 0.007).

In a multivariate analysis considering the non–cell-type variables patient age, HLA compatibility, CMV status, and female donor to male recipient (n = 5), only the {gamma}{delta}TCR+ T-cell dose remained significantly (P = 0.002) associated with aGVHD when including all variables in the analysis. The CD34+ cell dose was significant only when excluding Tregs from the analysis (P = 0.026). However, the Treg cell dose was not significant in the multivariate analysis (P = 0.35) even after excluding the CD34+ cell dose from the analysis (P = 0.11).

We then evaluated whether high doses of {gamma}{delta}TCR+ cells had a cumulative effect when combined with low doses of Treg cells. To prove this hypothesis, we considered only those patients who received a graft containing either a Treg cell dose below and a {gamma}{delta}TCR+ cell dose above median (group 1) or vice versa (group 2). Thirty-three patients were included in this part of our analysis. Among the 16 patients within group 1, 2 developed aGVHD grade 0-I, whereas 14 (88%) developed aGVHD grade II-IV. In group 2 (n = 17), 11 patients developed aGVHD 0-I, whereas only 6 (35%) patients developed aGVHD II-IV, which is statistically significant compared with group 1 (P = 0.004). This supports the notion that high doses of {gamma}{delta}TCR+ cells and low doses of Treg cells seem to have a cumulative effect with regard to the severity of aGVHD. Among the non–cell-type variables, positive recipient CMV status seemed to be associated with aGVHD II-IV (P = 0.03).

Chronic GVHD. Eleven patients were not evaluable for chronic GVHD (cGVHD) because of death before day 100. From a total of 52 patients, 12 (23%) developed no cGVHD, whereas limited cGVHD was seen in 16 (31%) and extensive cGVHD in 24 (46%) patients. When comparing patients who developed no cGVHD with patients developing either limited or extensive cGVHD, no significant association between cell-type variables and cGVHD could be observed (data not shown).

Treatment-related mortality. TRM including mortality from infection (n = 10) was observed in 19 patients (30%). We could not document any significant difference in the cumulative incidence of TRM with respect to the number of infused T-cell subsets as well as CD34+ cells.

Relapse and overall survival. Among 63 patients, 17 (27%) suffered a relapse of their underlying disease. Statistical analysis of the cell-type variables did not show any significant correlation between the cell subsets investigated in our study and relapse (data not shown). Currently, 27 patients (42%) are still alive with a median follow-up of 30.1 months (range, 0.6-62). Among cell-type variables, none of the cell subsets significantly affected overall survival (data not shown).


   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
Because the success of PBSCT for hematologic malignancies can still be undermined by GVHD, further investigations elucidating how graft composition affects GVHD incidence are needed. In our study, we focused on the influence of Tregs and {gamma}{delta}TCR+ T cells on GVHD in the setting of unrelated PBSCT after intensive conditioning therapy. The data clearly suggest an unrevealed association between the infused number of these T-cell subsets and the outcome for patients. Suppressive properties of murine and human-derived Tregs have clearly been shown in several studies (21, 24, 25), and their ability to prevent GVHD after HSCT has been well investigated in murine models (26). In addition, the desired graft-versus-tumor effect does not seem to be abrogated by Treg function (10). Recent studies of related PBSCT in patients also revealed an inverse correlation between Tregs in grafts and later in peripheral blood of recipients on the extent of GVHD (11, 27). Up to now, their role in the unrelated setting has not been defined. Here, we have observed an association between higher counts of Tregs in the graft and a reduced incidence of aGVHD II-IV in the respective unrelated recipient, which is consistent with the results by Rezvani and Miura (11, 27). Additionally, the CD4+CD25+ cell dose inversely correlated with aGVHD II-IV as well, which is in contrast to recent studies of related transplantation demonstrating an insignificant (11) or even positive correlation (12). These differences may be at least partly related to different transplant protocols, including T-cell depletion in one study, or to patient characteristics. Another explanation could also be that in our cryopreserved PBSC grafts, the number of recently activated T cells also expressing CD25 without having suppressive properties was low, so that CD4+CD25+ and CD4+foxp3+ in this case represented a comparable population. Unlike Zorn (28), who determined the frequency of Tregs after HSCT, we could not see a significant association between Tregs and the extent of cGVHD, which suggests that the content of Tregs in the graft is more important for the development of aGVHD, whereas cGVHD is affected by other factors, including Tregs generated or expanded in vivo. In the multivariate analysis, however, Treg cell dose remained insignificant, even after excluding CD34+ cells from the analysis. Because the heterogeneity of patient characteristics possibly interferes in the multivariate analysis, a larger study with a more homogeneous group of patients seems to be warranted.

We further investigated the relationship between {gamma}{delta}TCR+ T cells in PBSC grafts and the incidence of clinical GVHD. Despite the variety of characteristics attributed to {gamma}{delta}TCR+ T cells, their exact role in the immune system remains unclear. Murine models have led to the conclusion that {gamma}{delta}TCR+ T cells play a critical role in GVHD, but the results reported thus far do not concur. Whereas Drobyski et al. (18) postulated a protective effect of {gamma}{delta}TCR+ T cells when {alpha}ßTCR+ T cells were infused 2 weeks after bone marrow transplantation, Blazar et al. (15) showed that the infusion of donor {gamma}{delta}TCR+ T cells induced lethal GVHD in mice, and Maeda et al. (29) observed similar effects of host {gamma}{delta}TCR+ T cells. Anderson et al. (30), however, did not find any correlation between host {gamma}{delta}TCR+ T cells and GVHD in mice. In our study, the overall CD3+ cell dose did not show any significant association with GVHD, TRM, relapse, or survival being consistent with previous studies (4, 8, 31, 32). Interestingly, we detected a significant association between an increased donor {gamma}{delta}TCR+ T-cell dose and the cumulative incidence estimates of aGVHD grade II-IV, concurring with Maeda and Blazar (15, 29), which was proven even in a multivariate analysis. When correlating {gamma}{delta}TCR+ T cells with Tregs to exclude any interaction between the two variables, no significant correlation could be observed, which strengthens our results. Although the exact molecular mechanisms need to be further investigated, we assume that {gamma}{delta}TCR+ T cells are involved in mediating GVHD, possibly by direct cytotoxic effects (33) or by serving as antigen-presenting cells, as proposed by Brandes (34), or by a combination of both mechanisms. When evaluating a possible cumulative effect of Tregs and {gamma}{delta}TCR+ T cells, we could show in fact that low Treg doses and high {gamma}{delta}TCR+ T-cell doses seem to have a cumulative effect on the severity of aGVHD, which supports our previous results. Further studies discriminating between the different subtypes of {gamma}{delta}TCR+ T cells especially the V{delta}1 cells, located predominantly in the intestine (35), could be useful to elucidate their role in GVHD. Additionally, given their unique properties, they might also mediate a graft-versus-leukemia effect. In fact, we could not show such an association, which again might be a result of the heterogenous patient population.

The role of CD34+ cells transfused within the graft is still under investigation and seems to depend on particular variables. In fact, our study is the first to show the impact of CD34+ cell dose in the setting of unrelated PBSCT after intensive conditioning. Interestingly, the results do not agree with the data published for related transplants (4, 5, 7, 31, 32), i.e., higher CD34+ cell dose correlated with a reduced risk of aGVHD. However, there was a significant correlation between CD34+ and Treg cells possibly explaining the fact that the CD34+ cell dose only remained significant in the multivariate analysis when excluding Treg cells from the analysis. It is, of note, that all of our patients received an ATG-free regimen with uniform GVHD prophylaxis. Nevertheless, we cannot rule out a distinct influence of the type of conditioning on the results of our study. We could not see a significant association between CD34+ cell dose and cGVHD, relapse, TRM, or survival, being consistent with most of the studies (7, 8, 31).

Many risk factors unrelated to graft composition, such as patient age, HLA compatibility, sex mismatch, CMV status, donor parity, and stage of disease (35–37) are predictive for the development of GVHD. Nevertheless, most of these data were obtained in the setting of bone marrow transplantation and not PBSCT. Although Zaucha et al. showed patient age and CMV status to have an impact on aGVHD as well as age, sex, and stage of disease on cGVHD (3), Przepiorka and Dhedin were not able to see such an association after PBSCT (31, 32). In our study, only CMV status showed significant association with aGVHD, whereas HLA, age, and sex mismatch remained insignificant. One might argue that this might be due to the high rate of HLA compatibility in our study population as obtained by high-resolution typing.

In summary, our findings suggest a relationship between graft composition and clinical outcome of patients receiving allogeneic PBSCT from unrelated donors. Further studies are needed to support these observations, which might flow on into new strategies of graft manipulation or clinical-scale adoptive transfer of T-cell subsets (38), to minimize risks of clinical stem cell transplantation.


   Acknowledgments
 
We thank Christian Thiede, Uta Oelschlägel, and Ines Wagner for excellent technical assistance with PCR and flow cytometry, respectively. Special thanks to Kristina Hölig and her team for performing apheresis procedures in all donors and to Verona Schwarze, Anja Maiwald, and Diana Doehler for excellent technical assistance. We are grateful to Michelle Meredyth-Stewart and Marc Schmitz for critically reading the manuscript and Cathrin Theuser for performing statistical analysis.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 http://ctep.cancer.gov/reporting/ctc.html Back

Received 10/27/06; revised 1/25/07; accepted 2/21/07.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Platzbecker U, Thiede C, Fussel M, et al. Reduced intensity conditioning allows for up-front allogeneic hematopoietic stem cell transplantation after cytoreductive induction therapy in newly-diagnosed high-risk acute myeloid leukemia. Leukemia 2006;20:707–14.[CrossRef][Medline]
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