We have investigated the immune status of patients with hematologic malignancies treated with a low intensity conditioning in preparation for allogeneic stem cell transplantation. Conditioning consisted of fludarabine, anti-T lymphocyte globulin and low-dose busulfan, followed by infusion of allogeneic blood stem cells. This protocol resulted in rapid engraftment and complete replacement of host with donor hematopoietic cells. Immunological parameters of these patients were compared to those patients who were conditioned by an aggressive myeloablative regimen. Distribution of cell surface markers of lymphocyte subsets from both groups of patients was similar, but different from that of normal control cells. Reduced intensity or non-myeloablative conditioning prior to allogeneic stem cell transplantation (NST), hardly lowered the normal T cell-dependent mitogenic response even during the early period following transplant, while the myeloablative treatments resulted in a suppressed mitogenic reaction and in slow immune recovery. Reactivity of non-MHC restricted cytotoxic T cells was also at a normal level in patients who were treated with NST. We conclude that stem cell engraftment following reduced conditioning may result in early reconstitution of immune responses assessed in vitro. We hypothesize that clinical application of NST may lead to faster development of effective immune responses against residual host-type malignant and abnormal non-malignant hematopoietic cells, although the role of fludarabine on post-transplant infections remains to be investigated in a larger cohort of patients. Bone Marrow Transplantation (2001) 28, 243–249.
High-dose chemoradiotherapy is generally administered as part of the conditioning regimen prior to allogeneic bone marrow transplantation (BMT) for various hematologic malignancies and non-malignant indications including genetic disorders.1,2,3,4 The general concept is to give the highest tolerable dose in order to reduce the tumor load to a minimum or to eliminate all abnormal host-derived hematopoietic cells, as well as to eradicate all immune cells of host origin in an attempt to prevent graft rejection.
Following BMT, patients suffer from systemic toxicity and an increased susceptibility to early and late fatal bacterial, viral and fungal infections, initially caused by the aplastic nadir and subsequently due to impaired immunological responses that may be related to the aggressive myeloablative conditioning pre-transplantation and/or mandatory drugs (eg cyclosporine A, methotrexate and corticosteroids) given as graft-versus-host disease (GVHD) prophylaxis.5,6,7,8 For a number of months and lasting up to 1 year following uneventful BMT, the immune system of patients is characterized by impaired immunological responses to recall antigens as well as to mitogenic or allogeneic stimuli.9,10 Designing a less intense conditioning regimen in which engraftment is achieved without compromising the anti-tumor effect, is of great importance to reduce the well-known complications post myeloablative stem cell transplantation.
We recently designed a protocol consisting of fludarabine, anti-T lymphocyte globulin (ATG) and low-dose busulfan, which lowered the intensity of the conditioning regimen to the range of a non-myeloablative treatment pre-stem cell transplantation (NST), and induced intensive, but transient immunosuppression. It allowed engraftment of allogeneic stem cells as well as elimination of tumor and/or abnormal host cells in patients with hematologic disorders and in patients with non-malignant diseases requiring stem cell transplantation.11
Fludarabine is a cytotoxic analogue of deoxyadenosine which induces complications less frequently than other common cytotoxic drugs,12,13 and was shown to induce remissions in patients with B cell chronic lymphocytic leukemia and follicular lymphoma.12,14
Fludarabine was also administered in combination with other cytotoxic agents as part of a non-myeloablative conditioning, for patients who were ineligible for conventional myeloablative protocols prior to allogeneic BMT.15 Here, we evaluated several immunological parameters of patients conditioned either by a non-myeloablative or by an aggressive myeloablative protocol prior to allogeneic stem cell transplantation.
Materials and methods
Patient characteristics, including age, sex, race and disease are summarized in Table 1. Twenty-seven patients were conditioned with a myeloablative protocol (protocol I) consisting of cytoxan 60 mg/kg × 2 days (i.v.); total body irradiation (TBI), 1200 cGy given as 6 fractions of 200 cGy (19 patients received additional TLI 150 cGy × 4); six patients received additional VP-16 (Pharmachemie BV, Haarlem, The Netherlands) 1500 mg/m2 × 1 (i.v.); and seven patients received additional melphalan (Pharmacotherapie) 60 mg/m2 × 1 (i.v.)). A total of four patients was conditioned with busulfan (Pharmacotherapie) 4 mg/kg × 4, thiotepa (Pharmacotherapie) 5 mg/kg × 1 day and cytoxan 60 mg/kg × 2 days. All patients received bone marrow cells obtained from fully matched siblings. A second group of 10 patients was conditioned with NST consisting of fludarabine (Schering AG, Berlin, Germany) 30 mg/m2/day (Schering) for 6 days, busulfan 4 mg/kg/day for 2 days (p.o.) and anti-T lymphocyte globulin (ATG, Fresenius AG, Munich, Germany) 10 mg/kg/day (i.v.) for 4 days (protocol II) as previously described.11 Allogeneic hematopoietic blood stem cells were collected following mobilization with G-CSF (Neupogen 5 μg/kg twice daily (s.c.) for 5 days). Bone marrow and G-CSF-mobilized blood stem cells were used with no further in vitro manipulation. The sole prophylaxis against GVHD consisted of cyclosporine A 3 mg/kg starting from day −1. None of the patients received donor lymphocyte infusion post grafting. Patients with acute GVHD grade II or early chronic GVHD requiring immunosuppressive therapy, which could effect immunologic reconstitution, were not evaluated for their immune responses. All patients, upon entering the study, signed an informed consent form, which was approved by the Institutional Review Board.
An attempt to find differences in immune reconstitution related to the basic disease failed to show any pattern of distinction and therefore the data of all patients were pooled.
Peripheral blood mononuclear cells (PBMC)
PBMC were obtained from patients or from healthy controls. All cells were separated on Ficoll–Paque gradients as described.16
Cell surface markers were detected by indirect immunostaining using flow cytometry by a FACScan (Becton Dickinson, Mountain View, CA, USA). PBMC were incubated for 45 min at 4°C with the following monoclonal antibodies: anti-CD3, anti-CD8, anti-CD4, anti-CD56 (NKH-1), anti-CD16, anti-HLA-DR and anti-CD14 (Beckman-Coulter, Nyon, Switzerland and Pharmigen, San Diego, CA, USA). After two washes with phosphate-buffered saline containing 2% bovine serum albumin (Sigma, Rehovot, Israel) and 0.1% sodium azide, cells were resuspended in a solution of fluorescein-isothiocyanate-labeled F(ab′)2 fragment of sheep anti-mouse IgG (Sigma, Israel) for 45 min at 4°C. The cells were then fixed with 1% paraformaldehyde for subsequent analysis by FACS scan.
PBMC (105) were cultured in flat-bottom 96-microwell plates (Costar, Cambridge, MA, USA) with phytohemagglutinin (PHA, 0.625 μg/ml; Murex, Dartford, UK) in a total volume of 0.2 ml per well. Culture medium consisted of RPMI 1640 supplemented with 2 mM L-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin (Biological Industries, Beit Haemek, Israel) and 10% heat-inactivated (56°C, 30 min) human AB+ serum. Cultures were incubated for 48 h at 37°C in a 5% CO2 humidified incubator, pulsed for 16–18 h with 1 μCi methyl 3H-thymidine (5 Ci/mM, Nuclear Research Center, Israel) per well and harvested with a semiautomatic multiple sample cell harvester (Skatron Instruments, Lier, Norway). Radioactivity expressed as c.p.m. was measured in a Betamatic Liquid Scintillation Counter (Kontron, Switzerland). Results are presented as stimulation index calculated by the following formula: c.p.m. of cells + PHA/c.p.m. of cells only.
Induction of IL-2-activated killer (LAK) cells in vitro
PBMC at a concentration of 1 × 106 cells/ml were incubated in culture medium supplemented with 6% heat-inactivated AB+ serum and 6000 IU/ml IL-2 (Proleukin; EuroCetus, Amsterdam, The Netherlands) in 25 cm2 tissue culture flasks (Greiner Labortechnik, Germany) for 4 days at 37°C in a 5% CO2/humidified air incubator.
Measurement of cytotoxic activity
Cytotoxic activity was tested by a 51Cr-release assay as previously described.17 IL-2-activated PBMC were collected and tested against NK-sensitive erythroleukemia cells (K562) and NK-resistant Burkitt's lymphoma cells (Daudi) in serial four-fold dilutions starting at a 32/1 effector/target cell ratio. The test was performed in culture medium containing 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA). Cells were harvested by a set of harvesting frames and macrowell tube strips with a special harvesting device (Skatron Instruments, Lier, Norway). Cytotoxic activity was determined by calculating the percent of specific lysis and lytic units as described.18,19 The cytotoxic activity is presented here by lytic units calculated for 20% lysis/106 effector cells.
Statistical significance was determined by the standard two-tailed, unpaired, Student's t-test.
Phenotypic analysis of PBMC
Peripheral blood samples obtained from patients following allogeneic hematopoietic stem cell transplantation (HSCT) were analyzed for cell surface markers of T cell subsets (CD3, CD4, CD8), NK cells (CD16, CD56), B cells (CD19), monocytes (CD14) and activated cells (HLA-DR). The phenotypic analysis was carried out on cells from two groups of patients that differed in their conditioning pre-HSCT and a control group of healthy donors. Conditioning by the myeloablative (protocol I) or the non-myeloablative regimen (protocol II) resulted in a similar cell subset distribution when analyzed 1 and 3 months following transplantation (Table 2). In comparison with the control group, both groups of transplant patients had a low percentage of CD3+ cells, a marked decrease in CD4+ cells and an increase in CD8+ cells leading to an inverted cell ratio of CD4/CD8 (1/2–1/4) compared with PBMC from normal donors (2/1). Following transplantation, the number of HLA-DR+ cells was elevated in comparison with the number of cells carrying this surface marker in normal PBMC. Patients preconditioned by the myeloablative protocol had high numbers of HLA-DR+ cells when tested 1 month post allogeneic HSCT. However, the percentages were reduced in PBMC 3 months following allogeneic HSCT. Percent of CD56+ and CD16+ cells was higher following HSCT than in normal controls excepting the reduced numbers observed 3 months following conditioning with the non-myeloablative regimen. The proportion of cells carrying CD19 or CD14 markers were comparable in PBMC from both groups of patients and not statistically different from the healthy controls. In summary, conditioning with aggressive myeloablation or by a less aggressive regimen did not lead to major differences in the distribution of peripheral blood cell subsets.
The in vitro T cell-dependent mitogenic response was determined for two groups of patients who underwent allogeneic HSCT. Results presented in Table 3 show that conditioning by the myeloablative protocol leads to suppressed PHA responses 1 and 3 months post transplantation (median stimulation index 4 and 8, respectively). This response, although increasing (median stimulation index 34), did not return to normal levels even in samples obtained 12 months post transplantation. On the other hand, PBMC samples, taken from patients given the non-myeloablative conditioning, reacted to a T cell mitogenic stimulus almost as well as normal control cells even in samples taken as early as 1 month following NST (median of stimulation index 96). This high response was maintained throughout a follow-up period of 1 year and was not statistically different (P > 0.05) from the response of normal cells (median of stimulation index 140). The response to another T cell mitogen, ie Con A, showed the same pattern as that of PHA (data not shown). We therefore conclude that following non-myeloablative regimen, patients retain an almost intact in vitro T cell-dependent proliferative mitogenic response.
The ability to induce non-MHC-restricted cytotoxic activity in vitro was determined in PBMC from patients conditioned with non-myeloablative regimen and the results were compared with those of PBMC from normal controls. Cytotoxicity was measured against labeled NK-resistant (Daudi) and NK-sensitive (K562) target cells. Results presented in Figure 1 show that at 1 month following transplant, cytotoxic activity induced against K562 cells was significantly higher (P = 0.02) than that observed in control cells, decreased with time and reached normal levels after 3 months. IL-2-induced cytotoxicity directed against Daudi cells was lower 1 month post transplant, but not significantly different from activity measured in PBMC from healthy controls (P = 0.42). Over time following transplantation, cytotoxicity was similar to that of the control cells. In summary, non-MHC-restricted cytotoxic activity in cells from patients conditioned with non-myeloablative treatment was retained or even enhanced, compared with normal healthy controls.
Clinical evidence of infection in recipients of bone marrow transplants following low intensity or myeloablative conditioning
We have assessed the number of infectious complications in recipients of allografts conditioned with myeloablative as compared to low intensity conditioning (Table 4). There are no obvious differences between the two groups of patients in the incidence of bacterial (33% vs 23%, P = 0.4), and fungal (0% vs 8%, P = 0.38) infections, nor in the incidence of Pneumocystis carinii (5% vs 0%, P = 0.62). There seems to be an increased incidence of cytomegaloviremia (CMV) (61% vs 24%, P = 0.03) in patients treated with low intensity fludarabine, compared to patients treated with myeloablative regimen. However, CMV patients conditioned with the low intensity regimen (protocol II) reflect positive PCR in routine blood samples taken mainly from asymptomatic patients, while the viremia in the myeloablative conditioned group represents positive viral cultures taken from febrile patients at a time when the PCR technique was not yet in use.
Patients treated by low intensity conditioning prior to allogeneic stem cell transplantation, featured early recovery of T cell-dependent mitogenic response and non-MHC restricted cytotoxicity. These results are in contrast to the impaired immune reactivities observed in patients conditioned by a standard aggressive myeloablative regimen, as described here and by others. It has been shown previously that T cell proliferative responses assessed by stimulation via the CD3 or CD2 pathways as well as by a non-specific lectin like PHA, were decreased for over 12 months post transplant compared with T cell responses of donors, serving as normal controls.7,20,21,22 The impaired T cell functions and the B cell-related reactivities23,24 of transplant recipients are thought to be mostly due to some unknown qualitative rather than quantitative defect in the circulating lymphocytes.5,8 Phenotypic analysis of PBMC demonstrated that within 3–6 months various cell subpopulations recover and most return to normal levels within 12 months post allogeneic transplantation.6,22,25,26,27 Following both myeloablative and low intensity conditioning, our FACS analysis of PBMC is in accordance with data published previously, showing a decrease in the absolute number of CD4+ cells accompanied by an increase in CD8+ cells which led to an inverted CD4/CD8 cell ratio.6,22,25,26,27 Both regimens resulted in a long-lasting increase in activated cells carrying the HLA-DR cell surface marker in contrast to the number of HLA-DR+ cells in normal PBMC. The presence of continuous allogeneic stimuli provided by the hematopoietic allograft is possibly the reason for the elevated number of activated cells as described also previously.25,28 In addition, following the two types of conditioning, we observed elevated numbers of CD56+ and CD16+ cells known for their natural killing activity against tumor cells.
After NST, the non-MHC-restricted cytotoxicity induced by IL-2 activation directed against NK-resistant target cells (Daudi) in vitro, was almost as high as that of normal PBMC, whereas the cytotoxicity directed against NK-sensitive target cells (K562) was even higher than that of normal controls, especially during the early period post transplantation. Although we did not test the killing activity induced in vitro following conditioning by the conventional aggressive myeloablative protocol, it has been shown that NK- and lymphokine-activated killing activities can be detected early following HSCT and might play an important role in promoting anti-tumor effects to eradicate residual malignant cells after transplant.29,30,31 It was important to establish that our low intensity regimen did not disrupt NK cell activity since NK cells may play a role in engraftment, in prevention of GVHD and in exerting graft-versus-tumor effects.31 We did not test the expression of IL-2R on cell surface PBMC from patients conditioned by the low intensity protocol. Nevertheless, the ability to induce killing activity by IL-2 stimulation, clearly indicates that these cells do bear IL-2R on their surface. As others have demonstrated following a conventional myeloablative regimen, expression of p55 and p75 chains of the IL-2R early after allogeneic transplantation was comparable to their expression in healthy control samples30 and the impaired T cell immune reactivity observed following the myeloablative protocol was presumably related to a functional inability to produce IL-232 and/or to activational defects in host-treated cells.33
The use of the purine-analog fludarabine in the context of NST may offer a marked benefit over alternative aggressive myeloablative bone marrow transplantation protocols. NST could be much more attractive if it could be confirmed that it is not accompanied by long-lasting impaired immune responses. Considering the potential long-term immunosuppressive effects of fludarabine, the data presented suggesting that rapid immune reconstitution follows NST in recipients of blood stem cells is indeed encouraging, since they are not disadvantaged in comparison with myeloablated recipients reconstituted with bone marrow allografts. Improved immunocompetence following stem cell transplantation may allow allogeneic lymphocytes to displace residual malignant or genetically abnormal host type hematopoietic cells more efficiently. Although the use of fludarabine in the setting of low intensity conditioning has been reported previously, only hematopoietic reconstitution and disease-free survival have been described,15 whereas we focus here on the immune status of patients following HSCT. In order to study the clinical relevance of the immune status of transplant recipients, we have monitored the incidence of invasive bacterial infections, CMV viremia, fungal infections and Pneumocystis carinii in the two groups of patients. Although larger cohorts of patients must be investigated in order to determine the potential advantageous or disadvantageous effects of fludarabine on the course of post-transplantation infections, our analysis shows that there are no significant statistical differences in the incidence of infectious diseases in the two groups of patients, except for CMV. However, it may well be that the lower incidence of viremia among historical controls of conventionally conditioned BMT recipients is due to the fact that sensitive PCR technologies were not available at that time. The number of patients is too small for a meaningful clinical evaluation of the role of fludarabine on the incidence of infectious complications post transplantation. Clearly, the immunological status of T lymphocytes in the patients is not the sole parameter that determines susceptibility or resistance to infections. The first line of defense is provided by polymorphonuclear cells and macrophages whereas T cell-dependent responses represent the first line of resistance against viral infections and other intra-cellular infections, as well as fungal infections. T cells may also affect resistance against infections by generating T cell-dependent antibody responses. However, some of the immune responses are T cell-independent. The precise role of T cell function on susceptibility and resistance to infections post transplantation may be difficult to assess because other factors such as GVHD, drugs that are required to prevent or treat GVHD as well as portals of entry of infections that may be created by the conditioning or by complications of the conditioning such as mucositis, may be difficult to monitor precisely. Furthermore, the effect of each of the components of the conditioning, especially fludarabine which is a relatively new agent in the field of bone marrow transplantation, on various cell subsets of the immune system other than T lymphocytes such as macrophages and dendritic cells should be a subject of separate investigation. Since fludarabine is the drug of choice for B type malignancies, its effect on B lymphocytes must also be taken into consideration as well as the net balance between the effect on T cells and on B cells, which is currently unknown. In future studies, the role of fludarabine in the generation of antibodies to recall antigens would also be of interest and of potentially important clinical relevance. In trying to compare the immunologic status of BMT and NST recipients, it should be remembered that transplants of blood stem cells contain a larger proportion of donor T lymphocytes, which may also contribute to improved T cell functions following NST. Hence, the advantage of reduced conditioning may be related in part to the quality of the graft rather than to improved conditioning. However, if one sees the overall benefits of NST in comparison to BMT, the conclusions are still relevant. The feasibility of induction of durable engraftment of stem cell allografts following non-myeloablative conditioning is not new. Stable bone marrow engraftment following non-myeloablative conditioning with fractionated TLI alone34,35,36,37,38 or one fraction of TLI 200 cGy and a single dose of cytoxan39 led to induction of permanent and specific transplantation tolerance to all donor alloantigens, including skin allografts across strong MHC antigen barriers in mice and rats as has already been described by us34,35,36,37,38,39 and confirmed by others.40,41,42
Of 10 patients similarly treated with the non-myeloablative regimen, only 2 were mixed chimeras containing both donor and recipient cells, while 8 patients were already full donor chimeras one month following transplant. The clinical outcome of the patients described here has been discussed elsewhere.11 Together with recently published data featuring consistent engraftment of donor stem cells following NST, it can be concluded that myeloablative conditioning may not be mandatory for consistent engraftment of fully matched donor stem cells.11,15 Overall, our approach may prove useful to treat large numbers of patients with malignant and non-malignant indications for allogeneic bone marrow or blood stem cell transplantation, including elderly individuals and patients with less than optimal performance status. Currently, we are enrolling additional patients to determine if the therapeutic benefits of reduced intensity regimen can be confirmed using larger cohorts of patients on a long-term basis. A prospective randomized clinical trial will be required for assessment of the potential benefits of NST in comparison with conventional BMT.
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This work was carried out in the Danny Cunniff Leukemia Research Laboratory. We wish to thank the Gabrielle Rich Leukemia Research Foundation; Baxter International Corporation; the German–Israel Foundation; the Cancer Treatment Foundation; the Szydlowsky Foundation; and Pauline and Jerry Silverstein for their continuous support of our ongoing basic and clinical research in cell therapy.
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Morecki, S., Gelfand, Y., Nagler, A. et al. Immune reconstitution following allogeneic stem cell transplantation in recipients conditioned by low intensity vs myeloablative regimen. Bone Marrow Transplant 28, 243–249 (2001) doi:10.1038/sj.bmt.1703118
- immune response
- allogeneic bone marrow transplantation (BMT)
- non-myeloablative stem cell transplantation (NST)
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