T-cell depletion prevents from bronchiolitis obliterans and bronchiolitis obliterans with organizing pneumonia after allogeneic hematopoietic stem cell transplantation with related donors

Abstract

Bronchiolitis obliterans (BO) and bronchiolitis obliterans organizing pneumonia (BOOP) are late-onset non-infectious pulmonary complications (LONIPCs) following allogeneic hematopoietic stem cell transplantation (HSCT). In the present study 10 of 197 conventionally prepared stem cell recipients developed BOOP after 365 days and 6 patients developed BO 333 days post-transplant. No BOOP or BO was diagnosed following T-cell depletion (p<0.05). Chronic GVHD was ascertained in all BOOP patients and appeared significantly (p<0,001) more frequent in the conventional transplant group. The data confirm a strong association between T-cell activity, chronic GVHD, BO and BOOP and point out the impact of T lymphocytes in the pathomechanism of BOOP.

Pulmonary complications develop in 30–60% of allogeneic haematopoietic stem cell transplantation (SCT) recipients and are considered as major cause of morbidity and mortality.1 The use of prophylactic antimicrobial agents changed the spectrum of pulmonary complications from infectious to noninfectious, in particular late-onset noninfectious pulmonary complications (LONIPCs).2 LONIPCs occur 3 months after transplantation in 10–15% of cases and are life-threatening complications which can significantly reduce quality of life.3,4 Although the classification of LONIPCs is undefined, the most common categorizations include bronchiolitis obliterans (BO), bronchiolitis obliterans organizing pneumonia (BOOP) and interstitial pneumonia (IP) or idiopathic pneumonia syndrome (IPS).5

BOOP is less common than BO with an incidence of 1–1.7% in allogeneic SCT recipients.5 Among SCT-recipients with histologic BOOP, 5-year survival was estimated at 33%.6 Acute GVHD affecting the skin and chronic GVHD involving the gut and oral cavity were found to be the most important risk factors for the development of BOOP following SCT and there is some evidence that alloreactive T-lymphocytes in the context of acute and chronic GVHD may play an important role.6 Recent reports suggest that mutations of the NOD2 and TLR-4 genes may influence the risk of severe acute GVHD after allogeneic SCT.7 Since NOD2/CARD15 is also expressed on bronchial epithelial cells the question was raised whether NOD2/CARD15 and TLR-4 variants are also involved in the development of BO or BOOP.

This retrospective single-center study aimed to evaluate the impact of in vivo and in-vitro T-cell depletion or mutations of the NOD2 and TLR-4 (Thr399Ile) genes on the post-transplant development of BO and BOOP.

Design and Methods

Two-hundred and eighty-one patients undergoing BMT (n=33) or PBSCT (n=248) from genotypic HLA-identical (n=242) or HLA-mismatched (n=39) sibling donors between January 1998 and December 2003 were consecutively included. Patients were divided into two treatment groups characterized by either conventional transplantation of PBSC or BM (group 1, n=197) or in vivo T-cell depletion by administration of anti-CD52-antibody followed by transplantation of an unmanipulated graft (n=14), or receiving a CD34-purified transplant (n=70) (group 2, n=84). The conditioning regimen consisted of cyclophosphamide (120 mg/kg body weight [BW]) in combination with total body irradiation (TBI) in 4 daily fractions of 2.5 Gy (n=239), or oral busulfan (BU) (1 mg/kg BW every 6 hours over 4 days) in combination with cyclophosphamide (120 mg/kg BW) (n=23). Treosulfan (Medac, Hamburg, Germany), 12–14 g/kg for 3 days was used with subsequent cyclophosphamide (120 mg/kg BW) application in 19 patients. Inclusion criteria for CD34-PBSCT is as previously described.8 Patients scheduled for CD34-PBSCT received a conditioning regimen with fractioned TBI, cyclophosphamide (120 mg/kg), and thiotepa (10 mg/kg BW) with anti-thymocyte globuline (ATG) (ATG-S, Fresenius, Bad Homburg, Germany) 10 mg/kg of BW for 4 days (n=70). GVHD prophylaxis consisted of a short course of methotrexate on days 1, 3, 6, and 11 in combination with continuous intravenous cyclosporine in group 1 patients (n=197). Patients with CD34-PBSCT received no further GVHD prophylaxis. For in vivo T-cell depletion alemtuzumab (Campath 1-H, 10 or 20 mg) was administered for 5 days followed by continuous intravenous cyclosporine application. Post-transplant T-cell add-backs were performed as previously described.8 Patient demographics and treatment characteristics are summarized in Table 1. CD34 cell selection was performed using the CliniMACS device (Milteny Biotech, Bergisch Gladbach, Germany), as previously described.8 Pre-transplantation histocompatibility testing of recipients and related donors generally consisted of low- and intermediate-resolution HLA-A, -B, -C, and high resolution HLA-DRB1 and DQB1 DNA-based typing as previously reported.9 The diagnosis of BO and BOOP was based on clinical presentation, chest radiography, computed tomography (CT) or high-resolution CT (HRCT), pulmonary function tests (PFT) and pathological findings according to Palmas et al.3 Patients were classified as having BO if a decline in the forced expiratory volume of 1 s (FEV1) to less than 80% of the predicted value and FEV1/vital capacity (VC) less than 70% was observed. If lung biopsies could not be obtained for a definitive histological confirmation of BOOP, the diagnosis was assumed by correlating PFT findings (restrictive defect and normal expiratory flow) with CT and HRCT findings (peripherally distributed patchy air space consolidation, ground-glass attenuation, nodular opacities) in conjunction with clinical features after exclusion of an infectious etiology by bronchoalveolar lavages (BAL). Acute and chronic GVHD was graded according to standard criteria.10,11 Single nucleotide polymorphisms 8, 12, and 13 of the NOD2/CARD15 gene were determined by a Taqman® protocol as published earlier by Hampe et al.12 and Holler et al.13 Genotyping for SNPs of TLR-4 were analyzed by a Lightcycler protocol using hybridization probes as published earlier by Hamann and co-worker.14 PCR and subsequent melting curve analyses were performed using the Lightcycler device and related software.

Table 1.Clinical and demographic profile and transplant related data of the patient groups.

Statistics

Cumulative estimates were calculated with the use of the Kaplan-Meier method. A proportional hazards Cox regression model was used to assess the independent effect of several covariates on the clinical endpoint development of BOOP.

Differences between time-to-event distribution functions were compared by a log-rank test (Mantel-Haenszel). Differences between the groups concerning disease stage, relapse, death, acute and chronic GVHD were evaluated with the use of the Fisheŕs exact or χ test.

Results and Discussion

The clinical features, demographic profile and transplant related data of patients are summarized in Table 1. Both patient groups were observed over the same time period with a median follow up of 30 months after transplantation. Patients of group one showed a significantly higher portion of advanced disease stages and HLA-nonidentical donors. This correspond to criteria used to assign patients to treatment groups. Bone marrow was transplanted more frequently in the conventional transplant group (group 1) compared to the T-cell depletion group (group 2). Group 1 patients presented a higher incidence of acute as well as chronic GVHD (p<0.001). Treatment related mortality in group 1 was 33.7 %±3.5%, significantly higher than the 17.2%±4.2% seen in group 2 (p<0.0025).

Among all hematopoietic stem cell recipients the incidence of BOOP was 3.6 % and of BO 2.1 %. BOOP occurred a median of 365 days after HSCT, and a BO median of 330 days post-transplant. Ten of the patients with a disease-free survival of more than 3 months after transplantation fulfilled the diagnostic criteria for BOOP and 6 for BO. The most common symptoms associated with BO and BOOP were dyspnea, dry cough and fever. Lung biopsies with consecutive histological verification of clinically suspected BOOP were obtained in 5 patients. All cases of BO and BOOP were observed in group 1 (p=0.036). The estimated probability for the development of BOOP 690 days post-transplant was 8.9%±2.7%, and for BO 3.3%±1.3%.

Clinical characteristic of the patients who developed BOOP or BO after HSCT are shown in Table 2. While the overall incidence of grades I and II acute GVHD was 46% for all transplant recipients, 90%–100% of the patients with BOOP or BO had grade I–II acute GVHD (p<0.01). Nine of the 10 patients with BOOP were male and all 10 BOOP patients received allografts from female donors. Although chronic GVHD was more common in BOOP and BO compared to the overall incidence (70%), differences did not reach statistical significance. BOOP and BO resolved or remained stable in 81% of all cases under treatment with bronchodilators, corticosteroids and immunosuppression. Two patients. one with BOOP and one with BO, died of respiratory failure due to pulmonary disease progression and one BO patient died of severe sepsis. All but one BOOP patient and all of the BO patients received peripheral blood stem cells as transplants.

Table 2.Clinical and demographic profile of the patients who developed BO and BOOP after transplant.

Among the patients with BOOP and BO, heterozygous NOD2 mutations occurred with a frequency of 6.3% in recipients for SNP11 and 6.3% in donors for SNP12 respectively. Calculated haplotype frequencies did not differ from controls of an earlier reported study.15 This resulted in an overall percentage of 88% pairs with unmutated SNPs at the patient and donor side. For TLR-4, only SNP Thr399Ile was shown to influence GVHD.15 Here no differences in the mutation rate were observed for patients with or without BO or BOOP, neither on patient nor donor side (data not shown).

Although the pathogenetic mechanisms for the development of LONIPCs are unclear there is some evidence that alloreactive T-cells have an impact since non-infectious lung complications were observed in the context of donor lymphocyte infusions.4 Cooke et al. suggested an association between lung injury and alloreactive donor lymphocytes in a murine model16 while Majeski et al. reported that respiratory reovirus1/L induction of intraluminal fibrosis used as an animal model of BOOP is dependent on T lymphocytes.17 In our present study, BO as well as BOOP only developed among patients transplanted following conventional myeloablative conditioning combined with standard immunprophylaxis while T-cell depletion apparently seemed to protect against late pulmonary complications which corroborates the assumption of alloreactive donor T-cells to be involved in the inflammatory processes of BO and BOOP.2,4,6 Clinical Improvement of BOOP following extracorporeal photopheresis was reported to implicate some clinical relationship between BOOP and chronic GVHD.18 Although we could not identify acute or chronic GVHD as independent risk factors for BO or BOOP in multivariate analysis, acute as well as chronic GVHD were observed significantly less frequent in the T-cell depletion group.

Because of the recently reported association of single nucleotide polymorphisms (SNP) of the NOD2/CARD15 and TLR-4 gene with increased acute GVHD severity and TRM,13,15 it was assumed that NOD2/CARD15 or TLR-4 variants may contribute to changes in host immunity, predisposing for BO or BOOP.7 Despite the small number of cases, our results suggest BO and BOOP to develop independently from mutations of the NOD2 and TLR-4 (Thr399Ile) genes.

It is interesting to note that BOOP occurred almost exclusively in male recipients who received PBSCT from female donors. All patients except one who developed BO and BOOP were transplanted with G-CSF mobilized and peripherally collected stem cells. This might imply that besides donor gender, the graft composition or graft type could also be relevant for the induction of late pulmonary complications. The influence of the graft origin on immunologic reactions after allogeneic transplantation was confirmed by the fact that, when compared with BMT, PBSCT is associated with a significantly increased risk for high grade acute GVHD and extensive chronic GVHD.19 In addition to GVHD, conditioning-related toxicity as a major contributing factor for BO and BOOP has also to be taken into consideration.20,21 Although in the present evaluation there is an apparent imbalance between the clinical features of the patient groups, T-cell depletion resulted in significantly less TRM. This was probably due to both a reduction in applied conditioning-related toxicity and effective GVHD-prophylaxis. So diminished acute toxicity in combination with delayed T-cell immune reconstitution8 might be the cause for the significantly decreased occurrence of late noninfectious pulmonary complications in patients treated with T-cell depleted transplant procedures. Further clinical and experimental studies are needed to identify risk factors for LONIPCs and to substantiate T-cell mediated immune mechanisms in BO and BOOP to improve clinical outcome after allogeneic SCT.

Footnotes

• Authors Contributions MD wrote the manuscript and designed the study; DWB designed and supervised the study; AHE and RT performed statistical analyses; MK, RP, CS contributed in data collection.
• Conflicts of Interest The authors reported no potential conflicts of interest.

• Received August 28, 2006.
• Accepted February 15, 2007.