Conventional allogeneic peripheral blood stem cell transplantation (PBSCT) in patients with pretransplant medical risk factors is associated with a much higher incidence of treatment-related complications compared with conditioning regimens of lower intensity.1,2,3,4 Among these risk factors, the following situations or combinations of adverse factors have to be considered: older patients' age, active fungal infections prior to transplantation or sepsis during previous therapies, comorbidity or organ failures, high cumulative doses of radiotherapy or chemotherapeutic drugs taking into account their toxicity profiles.5,6,7
Nonmyeloablative regimens of allogeneic PBSCT offer promising approaches for successful transplantation in patients with the above-mentioned risk factors, at the same time reducing mortality during the early post transplantation period.8,9 The principle of these conditioning regimens consists of radiotherapy or chemotherapy combined with enhanced immunosuppression to allow engraftment and to reduce the risk of graft failure due to remaining recipient lymphocytes.10
There are different established protocols for dose-reduced conditioning, most applied regimens being represented by the model established by Storb et al, and the protocol according to Slavin et al.11,12,13 The model established by Slavin is based on the combination of fludarabine, busulphan (8 mg/kg), and antithymocyte globulin (ATG) for pretransplant conditioning.12 Immunosuppression after transplantation consists of at least CSA or tacrolimus (FK506). This approach significantly differs from the model according to Storb and is only nonmyeloablative by definition: after conditioning by Slavin's protocol, patient haematopoiesis is almost destroyed by busulphan and chimerism of peripheral blood cells is not regularly observed. The lower risk of graft failure is balanced by an increased toxicity using this regimen compared to the protocol according to Storb. If graft rejection occurs, the period of aplasia is very long because of marked myelosuppression by Slavin's protocol. Therefore, patient haematopoiesis would need too much time to recover and these patients are highly threatened by infections without haematopoietic transplantation. Taken together, the protocol according to Slavin can be called 'reduced toxicity' (compared with conventional allogeneic PBSCT) or – the term we will use here – 'dose-reduced PBSCT'.
The study presented here aimed at the investigation of early development of different lymphocyte subpopulations in patients undergoing dose-reduced PBSCT according to Slavin. For this purpose, we determined lymphocyte subpopulations before starting therapy and at day +30 after transplantation. Recovery of lymphocytes being involved in such critical events as cytomegalovirus (CMV) reactivation and graft-versus-host reaction (GvHD) was not only compared with patients who received conventional preparative regimens.14,15,16,17,18 Furthermore, we compared the prevalence and severity of GvHD as well as CMV reactivation in both groups and formed subgroups to identify correlations between lymphocyte status and these events. A special focus of this study is represented by NK cell reconstitution and its potential role in GvHD and CMV reactivation.
Patients and methods
This retrospective analysis was approved by the institutional ethical review board of the university hospital of Friedrich Schiller University Jena (Germany). Informed consent was provided by all investigated patients.
Patients
In all, 27 patients (see Table 1) who had undergone dose-reduced allogeneic PBSCT between December 1998 and October 2002 and 24 patients who had conventional allogeneic PBSCT between January 1999 and September 2002 were included in this study. The median age of dose-modified patients was 49 years (range 21–61 years), while conventionally pretreated patients had a median age of 37 years (range 20–50 years).
Table 1 - Characteristics of patients treated with dose-modified or conventional hematopoietic stem cell transplantation.
Full table Preparative regimens
All patients of the dose-reduced group received the conditioning regimen according to Slavin consisting of fludarabine (30 mg/m2, day -10 to -5), busulphan (4 mg/kg, days -6 and -5), and ATG (10 mg/kg, day -4 to -1, FreseniusR).12 Most patients of the myeloablative group (19/24) were prepared with TBI (total 12 Gy, day -7 to -4) and cyclophosphamide (60 mg/kg, days -3 and -2) including two patients with extended regimens (plus thiotepa or etoposide). In case of reduced TBI (8 Gy), these patients additionally received fludarabine before transplantation. Two patients were conditioned with the combination of busulphan (4 mg/kg, day -7 to -4) followed by cyclophosphamide (60 mg/kg, days -3 and -2).
Prophylaxis against GVHD
All patients of both groups received CSA starting with 3 mg/kg at day -1 followed by adaptation according to serum level. Patients with GvHD risk factors (one HLA mismatch or unrelated donor) were additionally treated with MMF beginning at day +10.
Definition of CMV risk group and CMV surveillance
Patients were attributed to CMV risk groups according to previously defined criteria applied for myeloablative transplantations: low risk (donor and recipient serologically negative for IgG and IgM, calculated with 1), intermediate risk (only donor serologically positive, 2) and high risk (positivity of recipient, 3). In our transplantation unit, surveillance of CMV reactivation is performed by the detection of CMV using PCR in neutropenia or staining of CMV protein pp65 in leucocytes after engraftment.
Analyses of peripheral blood lymphocyte subpopulations
Briefly, before staining of peripheral blood cells using SimulSetR (Becton Dickinson, Heidelberg, Germany) erythrocytes were lysed and white blood cells (WBCs) were collected by centrifugation. After this, different subsets including isotype control were stained separately: isotype control, IgG1-FITC/IgG1-PE; T helper cells, CD3-FITC/CD4-PE; T suppressor cells, CD3-FITC/CD8-PE; B cells, CD3-FITC/CD19-PE; and natural killer (NK) cells, CD3-FITC/(CD16+CD56)-PE. Samples were analysed using a FACSCalibur cytometer (Becton Dickinson) using CellQuest software (Becton Dickinson).
Data description and statistical analyses
Absolute counts of each subset of lymphocytes/
l were calculated using absolute number of lymphocytes/l and percentage of the corresponding lymphocyte subpopulation. Furthermore, CD4 counts were indicated according to CDC (Centres for Disease Control) for patients with HIV infections: Stage 1 CD4 >500/l; Stage 2 CD4 200–500/l; Stage 3 CD4 <200/l. Statistical analyses were performed using standard t-test or 
2 test as indicated. Results were considered statistically significant in case of P-values
0.05.
Results
Time of engraftment and reconstitution of total (WBCs)
Engraftment of WBC was defined as the increase of WBC higher than 1000 leucocytes/l and more than 500 granulocytes/l. In patients who received conventional conditioning regimen engraftment of WBC took place at median day +15 (s.d. 2.9 days), whereas we observed WBC engraftment at median day +11 (s.d. 3.8 days) after dose-reduced preparative regimen. We documented the engraftment of platelets – defined by stable or increasing counts of platelets without further necessity of substitution – 2 days later in both groups: median day +16 (s.d. 11.5 days) and median day +13 (s.d. 6.3 days), respectively. At day +30 total WBC counts differed significantly depending on conditioning regimen: 4.8 Gpt/l (s.d. 4.0) after conventional vs 3.5 Gpt/l (s.d. 1.9) after dose-reduced regimen (P=0.01).
Development of CD4 cells, CDC stage of CD4, CD8 cells and CD4/CD8 ratios after transplantation
Figure 1 indicates the distribution of absolute CD4 counts (T helper cells) in peripheral blood at day +30 after allogeneic PBSCT in case of conventional vs dose-reduced preparative regimen. Absolute counts of CD4 at day +30 were significantly higher (P<0.001) when patients were pretreated using conventional protocols: 282/l CD4 cells (s.d. 231 CD4/l) vs 130/l CD4 cells (s.d. 113 CD4/l). In order to translate these CD4 counts to different risk groups, we formed three subgroups according to CDC classification developed for application in HIV patients (Table 2). Our data demonstrate that in the case of dose-reduced regimen CD4 cells are grouped significantly higher as compared to conventional conditioning protocols (P=0.005, 2 test). Median counts of peripheral CD8 (T cytotoxic cells) cells in contrast were comparable in both groups (see Table 3) but differed significantly too (P=0.04), with a favoured reconstitution after dose-reduced conditioning protocol. Accordingly, the observed mean CD4/CD8 ratios after conventional and dose-reduced conditioning were significantly different: 1.29 vs 0.35 (P<0.001).
Figure 1.
Distribution of absolute peripheral CD4 counts at day +30 after conventional (left) vs dose-modified (right) conditioning regimen. Absolute CD4 values (/l) are shown for each patient dependent on the applied protocol prior to allogeneic transplantation.
Table 2 - CDC stages of CD4 in peripheral blood after conventional vs dose-modified conditioning.
Full table Table 3 - Development of B cells, cytotoxic T cells (Tc) cells, and NK cells in peripheral blood after conventional vs dose-modified conditioning.
Full table Reconstitution of CD19, CD8 and NK cells until day +30
Table 3 shows the development of peripheral B cells (CD19), cytotoxic T cells (CD8) and NK cells (CD16/56 positive). Absolute B-cell counts at day +30 were very low independent of the therapy applied before transplantation (median of 9 vs 25/l) with a broad variability in both groups suggesting an advantage after dose-reduced regimen (P=0.09). As for repopulation of cytotoxic T cells (CD8) and NK cells (CD16/56), our data demonstrate a significant acceleration (P=0.04 vs 0.008) of CD8 and NK cell recovery after pretreatment according to the protocol established by Slavin et al.
Onset of acute GvHD and CMV reactivation
In patients who underwent conventional allogeneic PBSCT, we observed an onset of acute GvHD at median day 23 while CMV reactivation occurred later (median day 35). In patients who received the conditioning protocol according to Slavin et al, acute GvHD arose on median day 25 while the onset of CMV reactivation was documented at median day 40.
Development of GvHD and immune reconstitution
Clinically mild GvHD (no GvHD or GvHD grade I) as well as severe GvHD (grade II, III or IV) were seen in about half the patients receiving conventional conditioning or dose-reduced regimen (Table 4). In order to investigate composition of lymphocyte subsets not only for different conditioning therapies but also relative to these clinical events after allogeneic PBSCT, we analysed immune reconstitution separately for both GvHD subgroups (Table 4). CD4 and CD8 absolute counts and CD4/CD8 ratio do not differ significantly between patients with low grade and severe acute GvHD in both subgroups. Interestingly, after conventional and dose-reduced preparative regimens, we can demonstrate higher absolute NK cell counts in GvHD 0–I (P=0.02 vs 0.07).
Table 4 - Correlation between acute GvHD and different lymphocyte subgroups after allogeneic PBSCT: conventional vs dose-modified conditioning.
Full table Correlation between immune status and CMV reactivation
In both conditioning groups, we observed a similar percentage of reactivation of CMV (conventional 25% vs dose-reduced 37%) considering the tendency with regard to differences of CMV risk groups (P=0.09, 2 test). While after conventional therapy CMV reactivation strongly correlated with CD4/CD8 ratio after engraftment (P<0.001, see Table 5), there is only a slight difference between both subgroups in dose-reduced regimen.
Table 5 - CMV reactivation dependent on immune reconstitution after allogeneic PBSCT: conventional vs dose-modified conditioning.
Full table In addition, we also investigated whether there is an influence of CMV seropositivity on immune reconstitution (CD4/CD8, NK absolute counts). Neither in patients who underwent conventional protocols nor after a dose-reduced conditioning regimen was one of these parameters affected by the status of CMV seropositivity.
Incidence of CMV reactivation dependent on extent of acute GvHD
Table 6 shows the percentage of CMV reactivation as well as the median CMV risk group and CD4/CD8 ratios within both GvHD subgroups for each conditioning regimen. These data suggest a higher incidence of CMV reactivation in case of severe GvHD only in patients conditionally pretreated before allogeneic PBSCT. Furthermore, among patients receiving a conventional protocol a significantly lower CD4/CD8 ratio in case of CMV reactivation (P=0.02) can only be observed in patients with mild GvHD. A similar behaviour is suggested for grade II–IV GvHD after conventional conditioning (P=0.06). In contrast, in patients receiving dose-reduced pretreatment no characteristic CD4/CD8 ratios can be shown for CMV reactivation in grade 0–I vs II–IV GvHD (P=0.2 vs 0.3).
Table 6 - Incidence of CMV reactivation or primary infection dependent on extent of acute GvHD and development of CD4/CD8 ratios: conventional vs dose-modified conditioning.
Full table Discussion
Immune reconstitution after allogeneic transplantation is especially well described for bone marrow transplantation.19,20,21,22,23 Since the introduction of peripheral stem cell allografts an accelerated restoration not only of total WBC counts due to an earlier engraftment of granulocytes but also improved reconstitution of different subsets of lymphocytes can be observed.24,25,26,27 Beside this increase of absolute lymphocyte counts several reports could also demonstrate an improved reconstitution of lymphocytes on a functional level.28,29 After conventional conditioning prior to allogeneic PBSCT compared to bone marrow transplantation patients do not develop significantly more acute or chronic GvHD.4,30 In contrast, improved immune reconstitution following allogeneic PBSCT may prevent CMV persistence and CMV mediated pneumonia.31
To our knowledge, there are only a few data about the impact of the preparative regimen in allogeneic PBSCT on reconstitution of lymphocyte subsets post transplant and its importance for the development of GvHD or CMV reactivation. Most reports focus on patients who underwent allogeneic transplantation according to the Storb model.7,32,33 Shenoy investigated for the first time quantitative reconstitution of different lymphocyte subsets combined with functional assessment of these cells in allogeneic PBSCT, while Morecki described similar analyses after the preparative regimen according to Slavin.34,35 They could demonstrate an inverted CD4/CD8 ratio within the first year after transplantation as well as a good NK cell reconstitution at day +30 continuously decreasing in the following months. In recent reports on new approaches in nonmyeloablative protocols using Campath-1H, an increase in CMV reactivation combined with a delayed immune reconstitution leading to a lower incidence of acute GvHD is observed.36
Our data demonstrate that absolute CD4 counts and CDC stages differ significantly between both investigated preparative regimens. We can see peripheral CD4 counts after conventional therapy being higher than in dose-reduced treated patients. This observation cannot be explained by differences of WBC engraftment where the recovery of WBC is slower, but we suggest that the antithymocyte globulin applied in all cases of the dose-reduced subgroup may mediate the delayed CD4 reconstitution via its long-term effects. ATG is able to circulate for several weeks and could probably have a preference in binding to T helper cells.
The development of B cells is very slow after allogeneic PBSCT comparable with the situation after conventional chemotherapy and is not significantly affected by the extent of myelotoxicity before transplantation. Nevertheless, after dose-modified preparation there is a tendency of slightly better B-cell reconstitution but on a low median level. This difference could be due to an even more affected bone marrow microenvironment after high-dose chemotherapy, especially in combination with total body irradiation.
Soon after first reports on investigations of hematopoietic reconstitution after bone marrow transplantation NK cell development after transplantation became an important focus within this topic. In 1981, Dokhelar et al37 reported on fast restoration of NK cell activity after bone marrow transplantation and its stability when no infectious complications like CMV occurred. The early recovery of natural cytotoxicity after BMT could be confirmed by other groups.38 Several further works intended to investigate the differential potential and functional roles of NK cell activity in such fundamental events like GVHD, graft rejection, graft-versus-leukaemia (GvL) effects and CMV reactivation after allogeneic transplantation.39 Donor NK cells are attributed to improve donor engraftment and a potential role in GvL reactions without a necessary involvement in GvHD. Indeed, adoptive transfer of NK cells can increase GvL effects while reducing GvHD.40,41 Furthermore, NK cells are suggested to control viral reactivations like CMV in the case of deeply immunosuppressed patients. Recent repertoire analysis of killer activating receptors (KARs) and killer inhibitory receptors (KIRs) give first insights into the molecular mechanisms of NK cell-mediated GvHD and GvL effects and could provide a promising tool for NK cell-based strategies in allogeneic transplantation.42,43 In summary, these data reflect the variety of physiological functions as well as the immunologic and homeostatic role of NK cells in transplantation related events.44
Our data indicate that reduced toxicity grade before allogeneic PBSCT represented by dose-reduced conditioning protocol allows a better reconstitution of NK cells (CD16/56) and cytotoxic T cells (CD8) after transplantation. This may reflect a better GvL effect and/or an increased antiviral activity in these patients after engraftment. Interestingly, our data demonstrate a correlation between NK cell reconstitution and extent of GvHD in both subgroups. This may reflect the protective role of NK cells in GvHD development also after dose-reduced conditioning prior to allogeneic PBSCT.
The risk of CMV reactivation or disease after allogeneic PBSCT is not only decreased compared to BMT but also depends on the extent of the preparative regimen before transplantation.31 In detail, CMV reactivation can occur with a significant delay in patients who underwent allogeneic PBSCT according to the Storb model or increases significantly in patients receiving a dose-modified regimen that contains Campath-1H.33,36 Furthermore, the occurrence of GvHD can increase the risk for CMV reactivation after allogeneic PBSCT.
The onset of CMV reactivation in both subgroups of patients we investigated was comparable. The overall risk of CMV reactivation after both conventional vs dose-reduced conditioning before allogeneic PBSCT does not significantly differ (6 out of 24 vs 10 out of 27) if the distribution of CMV risk groups is considered (see Table 2). In contrast, there is a slight increase of CMV reactivation in patients with grade II–IV GvHD after conventional conditioning compared with grade 0–I GvHD within this pretreatment subgroup. The strongest protective parameter of CMV reactivation is reflected by CD4/CD8 ratio in conventionally pretreated patients. This parameter cannot be used after dose-reduced conditioning because of the described impaired CD4 reconstitution.
While dose-reduced patients of both GvHD subgroups belong to comparable CMV risk groups (average 2.4 vs 2.2), we observed almost identical rates of CMV reactivation (5/14 vs 5/13). Comparable CMV reactivation rates in these GvHD subgroups while there is no significant difference in absolute peripheral CD4 counts might be explained also by a highly impaired immune response in case of acute grade 0–I GvHD .
In order to evaluate the role of NK cells in a supposedly more sensitive manner, we formed further ratios of lymphocyte counts at day +30, for example, the NK/CD8 ratio (data not shown). Interestingly, in both conditioning groups there was a significantly higher NK/CD8 ratio when patients developed only mild acute GvHD. Furthermore, the NK/CD8 ratio was significantly higher in the subgroup of CMV-negative in comparison to CMV-positive patients being restricted to the protocol according to Slavin. In addition, after dose-reduced conditioning with similar CD4/CD8 ratios independent of CMV reactivation, not CD8 values but NK/CD8 ratios correlate with CMV status after transplantation. We are aware that the application of such ratios, having not been evaluated yet in larger series of patients, should be discussed very carefully. Nevertheless, these data let us hypothesise that after dose-reduced pretreatment NK cell reconstitution is not only faster but also that NK cells might play a more important role in virus defence than CD8 cells during the early post transplantation period. This may be due to substitution of the impaired development and function of necessary costimulatory cells like T helper cells by NK cells. We would therefore like to suggest the concept of additional lymphocyte ratios considering NK cell reconstitution to be introduced in further studies investigating immune reconstitution after allogeneic PBSCT.
Taken together, we could show differences in immune reconstitution of such specialised cells as CD4, CD8 and NK cells. Such differences obviously occur dependent on the intensity and grade of myeloablation of the applied preparative regimens. Furthermore, during early post transplantation period transplantation-related events – especially such as acute GvHD – correlate with defined constellations of lymphocyte subgroups. The facilitated NK cell recovery shortly after dose-reduced regimen according to Slavin might play an important role in the protection of CMV reactivation and inhibition of severe GvHD in this protocol.
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Acknowledgements
We are especially grateful to Professor M Boeckh (FHCRC, Seattle) for a critical reading of the manuscript and his helpful recommendations during the preparation. We also thank Ms Franke and Ms Herrmann for FACS analyses of peripheral blood lymphocytes and A Markusch for excellent help in analyses of patient data.
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