cd19+cd21low b cells and patients at risk for nih-defined chronic graft-versus-host disease with...

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TRANSPLANTATION

CD191CD21low B cells and patients at risk for NIH-defined chronicgraft-versus-host disease with bronchiolitis obliterans syndrome

Zoya Kuzmina,1 Katharina Krenn,2,3 Ventzislav Petkov,2 Ulrike Kormoczi,4 Roman Weigl,1 Arno Rottal,4 Peter Kalhs,1

Margit Mitterbauer,1 Lothar Ponhold,5 Gerhard Dekan,6 Hildegard T. Greinix,1 and Winfried F. Pickl4

1Department of Internal Medicine I, Bone Marrow Transplantation, 2Department of Pulmonology, 3Department of Anaesthesiology and General Intensive Care,4Institute of Immunology, 5Department of Radiology, and 6Department of Pathology, Medical University of Vienna, Vienna, Austria

Key Points

• B-cell subpopulationas biomarker forNIH-defined BOS.

Bronchiolitis obliterans syndrome (BOS), pathognomonic for chronic graft-versus-

host disease (cGVHD) of the lung, is a progressive and often fatal complication after

allogeneic hematopoietic cell transplantation (HCT). Biomarkers for the prediction and

diagnosis of BOS are urgently needed to improve patients’ prognosis. We prospectively

evaluated B-cell subpopulations and B-cell activating factor (BAFF) in 136 patients (46

BOS, 41 no cGVHD, 49 cutaneous cGVHD) to define novel biomarkers for early

diagnosis of National Institutes of Health-defined BOS diagnosed a median of 11 mo after HCT. Patients with newly diagnosed BOS

had significantly higher percentages of CD191CD21low B cells (25.5 versus 6.6%, P < .0001), BAFF (7.3 versus 3.5 ng/mL, P 5 .02),

and BAFF/CD191 ratio (0.18 versus 0.02 ng/103 CD191 B cells, P5 .007) compared with patients without cGVHD. The area under the

receiver operating curve for CD191CD21low B cells was 0.97 (95% confidence interval, 0.94-0.99) and a cutoff point >9% was optimal

for diagnosing BOS in patients with first drop of pulmonary function tests with a sensitivity of 96% and a negative predictive value of

94%. Thus, elevated levels of CD191CD21low B cells are a potential novel biomarker for HCT patients at risk for developing BOS at an

early stage and could allow improvement of patient outcome. (Blood. 2013;121(10):1886-1895)

Introduction

Chronic graft-versus-host disease (cGVHD) is a frequent and

serious complication of allogeneic hematopoietic cell transplanta-

tion (HCT) associated with high morbidity and nonrelapse mortality

(NRM), functional impairment, prolonged duration of immuno-

suppressive medication, and poor quality of life.1-4 Affected patients

present unique diagnostic clinical features as defined by the

National Institutes of Health (NIH)5 and most frequently the skin,

oral mucosa, eyes, and liver are involved.3,6 Bronchiolitis obliterans

syndrome (BOS) is the only single pathopneumonic manifestation

of cGVHD according to the NIH consensus.5 BOS is characterized

by the new development of airflow obstruction with a prevalence

ranging from 2% to 26% depending on the definition used.7-10

Lung biopsy specimens demonstrate bronchiolitis involving the

small airways and fibrinous obliteration of the lumen of respiratory

bronchioles and inflammatory cell infiltrates and variable degrees

of intralumental or peribronchiolar fibrosis.7 Currently, the cause

of BOS is still unknown and risk factors associated with BOS

include the use of peripheral blood (PB) stem cells, use of busulfan

or methotrexate (MTX) during transplant, intensity of the condi-

tioning regimen, impaired pretransplant lung function, low serum

immunoglobulin (Ig) levels, and respiratory tract infections during

the first 100 d after HCT.7,9,11-13 The clinical factor most commonly

associated with the development of BOS is the presence of

cGVHD in another organ.7,8,10,14 The NIH cGVHD consensus

project developed a definition of BOS in an effort to standardize

definitions for comparisons of data among research centers.5 A

subsequent modification of these criteria required absence of infection,

another manifestation of cGVHD, and forced expiratory volume

in 1 s (FEV1, ,75% predicted or decline .10%) and either signs

of obstruction as seen inFEV1/slowvital capacity,0.7 or obstruction in

the pulmonary function test (PFT) defined as residual volumeor residual

volume/total lung capacity .120% and signs of air trapping on

expiratory high-resolution computed tomography (HR-CT).5,7,10,15

The pathologic finding of obliterative bronchiolitis (BO) in lung biopsy

is considered to be a diagnostic feature of pulmonary cGVHD.5,7,10,15

The prognosis of BOS is poor, with an overall survival (OS)

of 44% at 2 y and 13% at 5 y.7,8,10,15 Despite advances in im-

munosuppressive treatment and supportive care, the dismal pro-

gnosis of BOS has remained unchanged. Better identification of

patients at risk for BOS and those who have developed very early

disease at a stage when the allo-injury may be more amenable to

immunosuppressive therapies could improve patients’ survival and

avoid irreversible organ damage. The early diagnosis of BOS

remains challenging. Because patients are asymptomatic in the

early stages, diagnosis is often delayed until significant airflow

obstruction has occurred, leading to dyspnea on exertion and poor

Submitted June 4, 2012; accepted December 20, 2012. Prepublished online

as Blood First Edition paper, January 9, 2013; DOI 10.1182/blood-2012-06-

435008.

H.T.G. and W.F.P. contributed equally to this study.

Presented at the Annual Meeting of the European Group for Blood and Marrow

Transplantation, Paris, France, April 2011 and at the Annual Meeting of the

American Society of Hematology, Orlando, FL, December 2010.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2013 by The American Society of Hematology

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exercise tolerance.5,7,15 No biomarkers for BOS have yet been

validated in large prospective cohorts.

B cells are increasingly assigned an important role in the

pathogenesis of cGVHD.16-19 Previously, we demonstrated abnor-

malities of B-cell function in patients with active cGVHD seen as

a distortion of B-cell homeostasis with an elevation of CD191

CD21low B cells and CD191CD21int-highCD38high IgMhigh transi-

tional B cells as well as an excess of B-cell activating factor

(BAFF).16,17 Furthermore, assessment of B-cell subpopulations

and BAFF/B-cell ratios in cGVHD patients allowed a distinction of

different impairments of humoral immunity seen as either

immunodeficiency or autoimmunity.17 BAFF is a cytokine of

the tumor necrosis family that plays an important role in B-cell

homeostasis20 and elevated BAFF levels correlated with the

occurrence of cGVHD and therapeutic response.18,21,22 Further-

more, genetic variation in recipient BAFF reportedly modulates the

phenotype of GVHD.23 Recently, Allen and colleagues24 observed

a heightened metabolic state and resistance to apoptosis in B cells

as well as ongoing signaling through the AKT and extracellular

signal-regulated kinase pathway, suggesting a mechanistic link

between increased BAFF levels, aberrant survival of B cells, and

disease pathogenesis in patients with cGVHD.

Here, we investigated B-cell subpopulations and plasma BAFF

levels as potential noninvasive biomarkers for diagnosis of BOS

and patients’ prognosis by analyzing cGVHD patients with NIH-

defined BOS in comparison with allogeneic HCT recipients

without cGVHD and patients with cGVHD of the skin without

lung involvement. Findings in newly diagnosed BOS patients were

confirmed in patients with refractory BOS.

Patients and methods

Patients and trial conduct

Since June 2005, all consecutive patients given an allogeneic HCT at our

institution and alive by d 100 were asked to participate in a prospective

study to investigate the incidence and severity of cGVHD according to

NIH-defined criteria and patient outcome.6 Patients gave written informed

consent in accordance with the Declaration of Helsinki. The study had the

approval of the Ethics Committee of the Medical University of Vienna. No

medical interventions were included in the study and transplant clinicians in

charge of the respective patients had no access to laboratory results. During

routine follow-up visits in the outpatient clinic, patients were assessed for

the presence of cGVHD, including physical exams and laboratory tests. All

patients had PFTs with arterial blood gas analysis prior to the start of

conditioning therapy and starting on d 100 after HCT every 3 mo for 3 y; in

the case of a decrease of FEV1 .10% compared with the value prior to

Table 1. Patient and transplant characteristics

All n (%) BOS n (%)

cGVHD skin

n (%) No cGVHD n (%) P

Number of patients 136 46 49 41

Median age in years (range) 41 (19-72) 37 (19-63) 43 (21-72) 46 (20-65)

Gender

Male 62 (46) 24 (52) 20 (41) 18 (44)

Female 74 (54) 22 (48) 29 (59) 23 (56)

Diagnosis

Acute leukemia 99 (71) 34 (74) 36 (75) 29 (70.7)

Chronic myeloid leukemia 6 (4) 3 (6.5) 3 (6) 0

Malignant lymphoma 16 (12) 7 (15.2) 4 (8) 5 (12.2)

Other† 15 (11) 2 (4.3) 6 (12) 7 (17.1)

Disease status at HCT

Standard risk‡ 61 (45) 25 (54) 15 (31)** 21 (51)** *.054

High risk 75 (55) 21 (46) 34 (69) 20 (49)

Conditioning regimens

Myeloablative 88 (65) 32 (70)* 38 (78) 18 (44)* *.018

Reduced intensity 48 (35) 14 (30) 11 (25)** 23 (56)** **.002

Stem cell donors

Related 34 (25) 11 (24) 13 (27) 10 (24)

Unrelated 102 (75) 35 (76) 36 (73) 31 (76)

HLA-identical 115 (84) 36 (78)* 40 (82) 39 (95)* *.02

HLA-mismatch 21 (16) 10 (22) 9 (18) 2 (5)

Stem cell source

Bone marrow 6 (4) 2 (4) 3 (6) 1 (2)

PB 128 (94) 43 (93) 45 (92) 40 (98)

Umbilical cord 2 (1) 1 (2.2) 1 (2) 0

Median number of CD341

cells 3106/kg transplanted (range)

6.5 (0.9-9.9) 6.5 (0.9-9.5) 6.5 (2.8-9.9) 6.5 (1.4-9.9)

Prophylaxis of acute GVHD

CSA 3 (2) 1 (2) 1 (2) 1 (2)

CSA 1 MTX 94 (69) 39 (85)* 37 (76) 18 (44)* *.0001

CSA 1 MMF 39 (29) 6 (13) 11 (22)** 22 (54)** **.003

CSA, cyclosporine A; MMF, mycophenolate mofetil.

*Indicates significant difference between the BOS cohort and the no-cGVHD cohort.

**Indicates significant difference between the cGVHD skin cohort and the no-cGVHD cohort.

†Other diagnoses included myelodysplastic syndrome and chronic lymphocytic leukemia.

‡Standard risk was defined as acute leukemia in first or second complete remission or chronic myeloid leukemia in first chronic phase. High risk disease included

myelodysplastic syndrome and advanced stage of acute and chronic leukemia.

BLOOD, 7 MARCH 2013 x VOLUME 121, NUMBER 10 B CELLS IN BOS 1887




HCT, HR-CT of the lungs and bronchoalveolar lavage (BAL) with

transbronchial biopsy were performed.

So far, a total of 188 patients have been enrolled in this study. Here, we

compared the subset of 46 patients developing BOS (supplemental Table 1) to

41 patients without evidence of cGVHD and 49 with cutaneous cGVHD

without lung involvement enrolled during the same study period. The patient

and transplant characteristics are shown in Table 1. Five patients (nos. 16, 20,

23, 25, and 31 in supplemental Table 1) had impairment of PFTs prior to HCT

(FEV1 , 75%). Twenty-one patients with BOS were previously reported.17

Besides clinical assessment, PB samples were analyzed by flow

cytometry on d 100 after HCT, at onset of cGVHD, at onset of pulmonary

involvement, and thereafter for up to 3 y. Serum Ig and BAFF levels were

serially analyzed at the same time points and correlated with the severity of

cGVHD. In patients without cGVHD, PB samples for analysis of B-cell

subpopulations and BAFF levels were obtained from d 100 after HCT every

3 mo for 2 y.

Transplant regimens

All patients underwent HCT according to standard-of-care or institutional

review board-approved protocols. Significantly more patients with BOS had

received myeloablative conditioning (P 5 .018) and GVHD prophylaxis

with cyclosporine A (CSA) and MTX (P 5 .0001) compared with patients

without cGVHD. Significantly more patients with an HLA-identical stem

cell donor were in the no-cGVHD cohort (P 5 .019). Significantly more

patients in the no-cGVHD cohort had a standard risk disease (P 5 .054),

received reduced-intensity conditioning (P 5 .002) and had GVHD

prophylaxis with CSA and mycophenolate mofetil (MMF) (P 5 .003)

compared with cutaneous cGVHD. GVHD prophylaxis consisted of CSA

with MTX in all patients given myeloablative conditioning and CSA with

or without MMF after reduced-intensity conditioning. All patients received

antiinfectious prophylaxes according to institutional guidelines.

Acute GVHD was graded weekly until d 100 after HCT and thereafter

every 2 to 3 wk.25 Chronic GVHD was graded according to the NIH

consensus criteria5 by assessing patients during routine clinical visits in the

outpatient clinic (Table 2).

PFT

All PFTs were performed at the Department of Pulmonology in accordance

with the guidelines of the American Thoracic Society and the European

Respiratory Society task force on standardization of lung function

testing,26-28 using the Autobox DL 6200 (Sensor Medics, Vienna, Austria).

All diffusion capacity of the lung for carbon monoxide (DLCO)

measurements were corrected for hemoglobin values obtained at the

time of determination of the diffusion capacity.28 All PFT values, except

the FEV1/vital capacity ratio, were expressed as a percentage of predicted

values and categorically assessed.

In accordance with NIH recommendations, the lung function score (LFS)

was calculated according to FEV1 and DLCO, each of which was categorized

as follows: 15>80%, 25 70% to 79%, 35 60% to 69%, 45 50% to 59%,

55 40% to 49%, and 65,40%.10,15 The scores for FEV1 and DLCO were

then summed and categorized as 0-3 according to NIH recommendations as

follows: LFS 2 5 category 0 (normal), LFS 3-5 5 category 1 (mildly

abnormal), LFS 6-9 5 category 2 (moderately abnormal), and LFS 10-12

5 category 3 (severely abnormal). BOS was defined according to modified

NIH criteria.5,7,10,15 In the case of missing histological proof of BO, at least

one other distinctive manifestation of cGVHD in a separate organ system

was required. In cases of decreased PFT, all patients had a routine work-up

to exclude respiratory tract infections, including CT scans, microbiologic

and fungal cultures from BAL, and viral screen.

Treatment of BOS

As soon as a diagnosis of BOS was established, all patients received

immunosuppressive therapy with methylprednisolone at 0.5 to 1 mg/kg.

Additional treatment consisted of calcineurin inhibitors in 28 patients and/

or extracorporeal photopheresis in 10 patients. All patients were evaluated

in 2- to 4-wk intervals during treatment of BOS. If no improvement

after 3 mo or progressive disease after 1 mo was observed, the systemic

immunosuppressive therapy was changed. Patients received a median of 2.5

(range, 1-7) treatment regimens for BOS. Salvage immunosuppressive

therapy of steroid-refractory BOS patients included extracorporeal photo-

pheresis (n 5 17), sirolimus (n 5 10), MMF (n 5 6), tacrolimus (n 5 10),

rituximab (n 5 5), and imatinib (n 5 6). The majority of patients received

topical treatment consisting of an inhalative steroid and salmeterol as

a long-acting bronchodilator. All patients were given additional immuno-

modulatory treatment with a macrolide in the course of disease.29

Isolation of blood cells, immunophenotyping, and

flow cytometry

Immunophenotyping of whole blood cells was performed as reported.16

We used optimal concentrations of the directly conjugated monoclonal

antibodies CD3-APC (S4.1), CD4-FITC (VIT4), CD8-PE (VIT 8), CD10-APC

(H110a), CD14-PE (MEM-18), CD16-PE (3G8), CD19-FITC (SJ25-C1),

Table 2. Characteristics of patients with BOS

At onset of BOS

(n 5 46)

At maximum

grade of BOS

(n 5 46)

n (%)

NIH-defined cGVHD lung severity score

1 25 (54) 16 (35)

2 18 (39) 21 (45)

3 3 (7) 9 (20)

LFS

0 (, 3) 1 (2) 0

1 (3-5) 11 (24) 9 (20)

2 (6-9) 31 (67) 28 (60)

3 (10-12) 3 (7) 9 (20)

Median LFS, range 7 (2-10) 8 (4-12)

Lung symptoms scale*

0 37 (80) 14 (30)

1 9 (20) 21 (46)

2 0 8 (17)

3 0 3 (7)

Median FEV1, range 61 (36-75) 52 (16-75)

Patients with FEV1 decline .10% 42 (91) 46 (100)

Patients with FEV1 <55% 12 (26) 23 (50)

Median FEV1/SVC ratio, range 0.69 (0.41-0.94) 0.62 (0.22-0.88)

Patients with FEV1/SVC ratio ,0.7 25 (54) 28 (61)

Median RV %, range 136 (71-222) 145 (63-270)

Patients with RV .120% 32 (70) 35 (76)

Median RV/TLC ratio, range 137 (96-208) 136 (85-223)

Patients with RV/TLC .120% 32 (70) 32 (70)

NIH-defined HR-CT changes

Air trapping 35 (76)** 39 (85)

Bronchiectasis 2 (4) 7 (15)

Small airway thickening 25 (54) 36 (78)

Transbronchial biopsies

Constrictive bronchiolitis 23/36 (64)

Alveolitis 13/36 (36)

Median time from d 0 to BOS, mo, range 11 (3-30)

Median therapies, range 2.5 (1-7)

LFS, lung function score; RV, residual volume; SVC, slow vital capacity; TLC,

total lung capacity.

*Symptoms reported by the patients as published by Filipovich and

colleagues.5 This score distinguished between no symptoms, mild symptoms

(shortness of breath after climbing one flight of steps), moderate symptoms (shortness

of breath after walking on flat ground), and severe symptoms (shortness of breath at

rest, requiring O2).

**In 3 patients HR-CT scan of the lungs was not valuable due to poor patient

compliance.

Transbronchial biopsies were performed in 41 patients; however, valid diagnosis

of biopsy specimens was possible in only 36 patients.

1888 KUZMINA et al BLOOD, 7 MARCH 2013 x VOLUME 121, NUMBER 10




CD21-PE (BU32), CD38-FITC (HIT2), CD56-PE (MEM-188),

CD45-FITC (VIT200), CD27-FITC (VIT14), and appropriate isotype

controls for characterization of B, T, and NK cell subpopulations.

Three-color data acquisition was performed on a fluorescence-activated

cell sorter Calibur flow cytometer (Becton Dickinson, San Jose, CA).

The flow cytometric gating algorithm on circulating CD191 B cells is

shown in Figure 1. The subset of CD191CD21low B cells contains both

CD21negative as well as CD191CD21low B cells as defined.30

Assessment of BAFF by enzyme-linked immunosorbent assay

Patients’ plasma samples were examined for soluble BAFF by enzyme-

linked immunosorbent assay (R&D Systems, Minneapolis, MN) according

to the manufacturer’s instructions.

Assessment of PB Ig levels

Serum levels of IgG, IgM, and IgA were quantified by nephelometry (BNH,

Dade Behring, Stratham, NH). The normal values were: IgG, 700 to 1600

mg/dL; IgA, 70 to 400 mg/dL; and IgM, 40 to 230 mg/dL. In all patients

with elevation of Ig levels, standard serum protein electrophoresis was

performed to exclude oligoclonal B-cell reconstitution.

Statistical analysis

The study was designed as a prospective cohort study including 136

patients that were followed longitudinally. NRM was defined as any death

in the absence of the underlying malignancy. Cumulative incidence of

NRM was estimated by using the Kaplan-Meier method with adjustment for

relapse as a competing risk event.31 Relapse was defined as recurrence of

malignancy after achievement of complete remission or any progression of

malignant disease. OS was calculated from d 0 of HCT to the day of death

from any cause or last follow-up. Patients were censored at the date of last

contact. OS was modeled using Cox regression methods.

Statistical pairwise comparisons of B-cell subpopulations and BAFF levels

with 2 patient cohorts were made using the Student t test. Differences in

diagnostic parameters between BOS patients and those without cGVHD or

cutaneous cGVHD without lung involvement were assessed with Fisher’s exact

test for continuous values and Student t tests of association for categorical values.

Of note, PFT values and other covariates for BOS were considered as categori-

cal variables in the analyses. Areas under the curve (AUCs) and corresponding

95% confidence intervals (CIs) were computed nonparametrically.

Differences were considered statistically significant at a P value , .05.

The data were calculated using SPSS Version 17.0 (IBM, New York, NY).

Results

We prospectively compared 46 patients with BOS to 41

without cGVHD and 49 with cutaneous cGVHD without lung

involvement (Table 1) and analyzed clinical, cellular, and

plasma variables as potential biomarkers for the occurrence and

severity of BOS. Onset of cGVHD was observed a median of

143 d (range, 74-732 d) after allogeneic HCT consisting of

mild disease in 9, moderate disease in 34, and severe disease in

3 patients (supplemental Table 2). The most frequently affected

organs at onset of cGVHD were skin (89%), eyes (87%), oral

mucosa (76%), and liver (80%). Seventy percent of patients

had NIH-defined classic cGVHD and 33% had the progressive

onset type of cGVHD.

Clinical characterization of BOS

During the prospective and serial assessments of all patients, BOS

was diagnosed in 46 at a median of 11 mo (range, 3-30 mo) after

HCT (Table 2). The overall cumulative incidence of BOS at 3 and

5 y was 27% (95% CI, 20% to 34%) and 30% (95% CI, 22% to

38%), respectively. At the time of diagnosis of BOS, all patients

had other manifestations of cGVHD, including skin (n 5 28),

eyes (n 5 30), mouth (n 5 11), and liver (n 5 28) (supplemental

Table 2). In 15 patients (33%), lung involvement was already

present at the onset of cGVHD. At onset, 54% of patients had

NIH-defined mild lung involvement and the mean of total lung

capacity and DLCO were 96% (range, 77% to 119%) and 59%

(range, 34% to 89%), respectively. Only 9 patients (20%) had

respiratory symptoms at onset [lung symptom scale (LSS) 1]

compared with 70% at maximum grade of BOS. At onset,

distinctive signs of BOS in HR-CT were present in 35 patients

(76%).5 Among 36 patients with transbronchial biopsy suitable for

Figure 1. Flow cytometric gating algorithm on PB B-cell subpopulations in patients without cGVHD (no cGVHD) and BOS. Whole blood was analyzed by multicolor

flow cytometry. B cells as defined by CD19 positivity and side scatter characteristics (SSC) were further stained in various combinations for IgD, CD21, CD27, CD38, and

CD10. Numbers indicate percent of cells within respective gates.





assessment, 23 (64%) had histological confirmation of BO

(supplemental Tables 2 and 3).32

During a median follow-up of 35 mo (range, 11.6-66 mo) after

HCT, the maximum severity of pulmonary cGVHD according to

NIH lung scores was mild, moderate, and severe in 35%, 45%,

and 20% of patients, respectively. The median LFS at onset and

maximum of pulmonary cGVHD were 7 (range, 2-10) and

8 (range, 4-12), respectively. The median FEV1 values at onset and

maximum of BOS were 61% (range, 36% to 75%) and 52% (range,

16% to 75%), respectively.

Relative numbers of CD191CD21low B cells as potential

diagnostic biomarker of new onset BOS

Early diagnosis of BOS is clinically challenging, because

afflicted patients can be clinically asymptomatic for prolonged

periods of time and in symptomatic patients, differential diag-

noses such as infections and toxicities have to be kept in mind.

Thus, an objective biomarker for diagnosis of BOS could allow

timely clinical intervention and may lead to improved patient

outcome.

Serum IgG levels were significantly lower in newly diagnosed

BOS patients (533 vs 1172 mg/dL, P , .0001) compared with

ones without cGVHD. Comparison of different T-cell subpopula-

tions revealed significant differences solely in CD41 T cells

(23.6% vs 17.5%, P 5 .004) in univariate analyses.

Analyses of BAFF levels and B-cell subpopulations were

performed in 46 patients at first decline of PFTs in combination

with distinct changes in HR-CT of the lungs, and the results were

compared with those obtained in 41 patients without cGVHD

analyzed at the same time, a median of 11 mo after HCT

(Table 3). Patients with newly diagnosed BOS had significantly

higher BAFF levels compared with patients without cGVHD

(7.3 vs 3.5 ng/mL, P 5 .02). Absolute numbers (146 vs 317 3

106/L, P , .0001) as well as percentages (10.2% vs 19.7%, P ,

.0001) of CD191 B cells were significantly lower in cGVHD

patients at onset of BOS compared with those without cGVHD.

The BAFF/CD191B-cell ratio was significantly higher in the

newly diagnosed BOS cohort (0.18 vs 0.02 ng BAFF/CD191 3

103 B cells, P 5 .007) compared with patients without cGVHD.

Patients with newly diagnosed BOS demonstrated significantly

higher percentages of CD191CD21low B cells (25.5% vs 6.6%,

P , .0001) compared with the cohort without cGVHD

(Figure 2A). CD191CD21low B cells were comprised of cells with

a CD38high and CD38low phenotype. CD191CD21lowCD38high

B cells consisted mostly of transitional B cells with high IgM

expression levels, CD10 positivity, and CD27 negativity, whereas

the fraction of CD191CD21low CD38highIgMlow plasmablasts was

small. In some BOS patients, a CD191CD21lowCD38low B-cell

population was significantly expanded. Only one-quarter (22.1

6 9.8%) of these cells coexpressed CD27 but all were

CD10negative. Patients with BOS had significantly higher percentages

of CD38high (7.1% vs 3.8%, P 5 .003) and CD38low (11.6% vs

4.5%, P 5 .0004) CD191CD21low B cells compared with patients

without cGVHD (Figure 3).

Because 6 patients (nos. 1, 7, 12, 16, 22, and 30 in supplemental

Table 1) did not fulfill all the criteria of the modified NIH

definition7,15 but had BOS according to a pulmonary specialist’s

assessment and thus were included in our prospective study, we

compared these to 40 patients having NIH-defined BOS. No

significant differences in potential cellular and plasma bio-

markers were observed between NIH-defined BOS and other

BOS patients (Table 3). Therefore, all following analyses were

performed with the whole cohort of 46 BOS patients.

Receiver operating characteristic curves of these biomarkers

distinguished BOS from non-GVHD. The AUC for relative

numbers of CD191CD21low B cells was 0.97 (95% CI, 0.94-

0.99) and a cutoff point .9% was found optimal for diagnosing

BOS in patients with first decline of PFTs corresponding to a

sensitivity of 96% and a negative predictive value (NPV) of 94%

(Figure 2B). The AUC for the ratio of BAFF/CD191 B cells was

0.89 (95% CI, 0.79-0.98) and a cutoff point ,0.05 had

a sensitivity of 68% and a NPV of 70%. The AUC for BAFF

was 0.74 (95% CI, 0.58-0.90) and a cutoff point .3 ng/mL had

a sensitivity of 82% and a NPV of 73%, as shown in Figure 2B.

The AUC for relative numbers of CD41 T cells was 0.68 (95%

CI, 0.56-0.80).

BAFF and B-cell subpopulations in BOS and cutaneous cGVHD

We prospectively compared 46 patients with newly diagnosed

BOS to 49 patients with cutaneous manifestations of cGVHD

without lung involvement who were analyzed a median of 11.6 mo

(range, 3-21 mo) after HCT (Table 3). BAFF levels (7.3 vs 4.5 ng/

mL, P 5 .03), CD191CD21low B cells (25.5% vs 7.2%, P ,

.0001), ratios of BAFF/CD191 B cells (0.18 vs 0.05 ng BAFF/

CD191 3 103 B cells, P 5 .003) as well as serum IgG levels (533

vs 754 mg/dL, P 5 .03) were significantly different between the 2

cohorts. Thus, relative numbers of CD191CD21low B cells can

serve as potential diagnostic biomarkers for new onset BOS.

Table 3. Significant differences in cellular and plasma biomarkers between newly diagnosed BOS and no-cGVHD patients in univariate

analysis

All BOS NIH-BOS

Other

BOS

No

cGVHD

cGVHD

skin

P NIH-BOS vs

other BOS

P BOS vs

no cGVHD

P BOS vs

skin GVHD

Number of patients 46 40 6 41 49

BAFF, ng/mL 7.3 7.5 7.0 3.5 4.5 .63 .02 .03

CD191 B cells 3 106/L 146 141 178 317 231 .5 ,.0001 .05

CD191 B cells, % of lymphocytes 10.2 10.1 16.3 19.7 11.2 .14 ,.0001 .67

Ratio ng BAFF/CD191 3 103 B cells 0.18 0.19 0.15 0.02 0.05 .8 .007 .003

CD191CD21low B cells, % of CD191 B cells 25.5 26.4 16.5 6.6 7.2 .18 ,.0001 ,.0001

IgG, mg/dL 533 522 605 1172 754 .5 ,.0001 .03

CD41 T-cells, % of lymphocytes 23.6 23.2 26.7 17.5 24.4 .41 .004 .7

Patients with cGVHD had erythematous lesions alone (n5 37), erythematous lesions and moveable sclerosis (n5 4), moveable sclerosis alone (n5 2), or superficial and

deep sclerotic features (n 5 6). NIH-BOS, BOS defined according to the modified NIH criteria7,15; other BOS, patients not fulfilling all criteria of the NIH definition but having

BOS according to pulmonary specialist assessment.





Correlation of cellular biomarker with severity of BOS

No correlation between CD191CD21low B cells, BAFF, CD191

B cells, and ratios of BAFF and B-cell subpopulations and NIH-

defined severity scores of pulmonary cGVHD were observed.

However, asymptomatic patients with a drop in PFTs and NIH-

defined changes in HR-CT (LSS 0) could be distinguished from

patients without cGVHD due to significantly higher CD191

CD21low B cells (25.4% vs 6.6%, P , .0001) and ratio of

BAFF/CD191 B cells (0.15 vs 0.02, P 5 .01). Furthermore,

these patients had significantly lower serum IgG levels (535 vs

1172 mg/dL, P 5 .0001). Thus, clinically asymptomatic patients

with elevated numbers of CD191CD21low B cells and a distortion

of B-cell subpopulations already have newly diagnosed BOS and

can by biomarkers be distinguished from patients without cGVHD.

Distinction of BOS from respiratory tract infections

We prospectively compared 49 patients with BOS to 46 patients with

lung infection occurring a median of 12.6 mo (range, 3-31 mo) after

HCT (Table 4). In patients without cGVHD, no significant

differences in BAFF and B-cell subpopulations were observed when

comparing patients with or without respiratory tract infections.

Patients with cGVHDwithout lung involvement and respiratory tract

infection had significantly fewer CD191CD21lowB cells (12.35% vs

25.5%, P 5 .001), more CD191 B cells (220 vs 146 3 106/L, P 5

.04), and higher serum IgG levels (895 vs 533 mg/dL, P 5 .0007)

when compared with newly diagnosed BOS patients. Thus,

relative numbers of CD191CD21low B cells can distinguish

BOS from respiratory tract infections both in patients without

cGVHD as well as in extrapulmonary cGVHD.

Cellular biomarkers for prediction of BOS and during the

course of BOS

Analyses of B-cell subpopulations and BAFF levels on d 100

after HCT revealed no significant differences between patients

developing BOS at later time points and patients not experienc-

ing cGVHD. On d 100, CD191CD21low B cells (Figure 4) were

elevated in both patient cohorts (20.8% in BOS compared with

15.7% in the no cGVHD cohort, P 5 .18), confirming that the

CD21low transitional B cells are the first to leave the BM and

subsequently give rise to more mature B-cell subpopulations

during improved B-cell reconstitution after HCT.33 At 6 mo after

HCT, relative numbers of CD191CD21low B cells decreased to

13.6% and 9.6% (P 5 .16). Whereas in patients without cGVHD,

CD191CD21low B cells further decreased to 6.7%, patients with

newly diagnosed BOS showed a renewed increase of CD191

CD21low B cells to 25.5% and these remained elevated throughout

the course of BOS.

Confirmation of potential cellular and plasma biomarkers in

patients with long-lasting BOS

Significant differences in B-cell subpopulations and BAFF levels

observed between patients with newly diagnosed BOS and those

never experiencing cGVHD were confirmed by serial follow-up

analyses of BOS patients not responding to systemic immuno-

suppressive treatment and thus experiencing more severe and

long-lasting disease. Significantly elevated BAFF levels, BAFF/

CD191 B-cell ratios, and percentages of CD191 and CD191CD21low

B cells as well as CD41 T cells were observed in all patient cohorts

with long-lasting BOS compared with patients without cGVHD

Figure 2. (A) BAFF and CD191CD21low B cells at

onset of BOS compared with patients without

cGVHD (no cGVHD). Patients with newly diagnosed

BOS have significantly higher BAFF levels and percent-

ages of CD191CD21low B cells compared with the no

cGVHD cohort. Data are shown as box plots with

medians and ranges, and P values are indicated. (B)

Receiver operating characteristic curves for percent-

ages of CD191CD21low B cells (dotted line), the ratio of

BAFF/CD191 B cells (dotted and dashed line), and

BAFF (solid line). Individual receiver operating charac-

teristic curves are shown.





(Table 5). Of note, relative numbers of CD191CD21low B cells were

25.5%, 27.8%, 26.5%, 22%, and 25.6% in patients with newly

diagnosed BOS, FEV1 < 55%, histologically confirmed BO, LFS 3,

and LSS 2 and 3, respectively, and thus significantly elevated both at

onset of BOS as well as in patients with long-lasting BOS.

No prognostic impact of potential cellular and plasma

biomarkers on survival and NRM of patients with BOS

In our prospective study, the 5-y probability of OS of cGVHD patients

with BOS was 77% compared with 69% in patients without cGVHD

(Figure 5A). In univariate analysis, only patients with LSS 1 at onset

(44.4% vs 89.2%, P 5 .001) and LSS 3 at maximum (33% vs 93%,

P 5 .027) had significantly worse OS. Furthermore, patients with an

NIH-defined severe lung score at maximum had significantly worse

5-y OS (56% vs 87%, P 5 .021) compared with moderate and mild

disease. No PFT values at BOS peak had a significant impact on OS.

Nine patients died, including 3 (33.3%) of relapse, 4 (44.5%) of

BOS, and 2 (22.2%) of cardiovascular failure. The 5-y cumulative

NRM with relapse as a competing risk for all patients was 11%

(95% CI, 4% to 19%). It was 15% (95% CI, 4% to 27%) in patients

with BOS compared with 7% (95% CI, 2% to 18%) in patients

without cGVHD (Figure 5B). Besides a higher LSS at onset of

BOS (HR 5.5; 95% CI, 1.2-24; P 5 .025), no other variable

including cellular and plasma biomarkers had a significant

influence on NRM in univariate analysis.

The 3- and 5-y cumulative relapse rates with NRM as competing

risk for all patients were 14% (95% CI, 6% to 22%) and 30% (95%

CI, 11% to 49%). It was 7% (95% CI, 0% to 16%) and 23% (95%

CI, 0% to 54%) in patients with BOS compared with 21% (95% CI,

7% to 36%) and 41% (95% CI, 18% to 64%) in patients without

cGVHD, respectively.

Discussion

BOS is a serious complication of allogeneic HCT with a dismal

prognosis; the OS rate is 13% at 5 y.7-9,11,12,15 Unfortunately, the

survival and treatment of patients with BOS have not improved

over the last 20 y.12,34,35 Challenges to progress in the medical

management of BOS include little knowledge about pathogenesis

to direct effective therapies and delay in diagnosis, because

patients with lung involvement by cGVHD can remain asymp-

tomatic for prolonged periods of time. We serially performed PFTs

starting on d 100 after HCT and in cases of BOS diagnosis,

corticosteroid therapy was administered. The main emphasis of this

prospective study was on the early diagnosis of BOS, assuming

that detection of BOS at an early stage when the immunologic

injury of pulmonary tissues may be more amenable to treatment

may change the course of the disease’s natural history. This could

lead to improved patient outcome. Previous publications expressed

this hope; however, no prospective interventional studies have been

reported to confirm this assumption. 7,15 A recent survey revealed that

screening asymptomatic patients for early detection of pulmonary

cGVHD had only been performed by ;50% of HCT centers.36

We identified, to our knowledge, the first potential diagnostic

biomarker for BOS after HCT, namely CD191CD21low B cells,

which were significantly higher in patients with newly diagnosed

BOS compared with patients without cGVHD. A cutoff point.9%

Figure 3. CD38 expression of CD191CD21low B cells.

BOS patients have significantly higher percentages of

CD38high and CD38low B cells compared with patients

without cGVHD. PB mononuclear cells were stained

with monoclonal antibodies against CD19, CD21, and

CD38. Data are shown as box plots with medians and

ranges, and P values are indicated.

Table 4. Significant differences in cellular and plasma biomarkers between newly diagnosed BOS and patients with respiratory tract

infections in univariate analysis

Newly diagnosed

BOS

Infection and

cGVHD

Infection and no

cGVHD

P BOS vs infection

and cGVHD

P BOS vs infection

and no cGVHD

Number of patients 46 29 17

BAFF, ng/mL 7.3 4.6 3.7 .08 .03

CD191 B cells 3 106/L 146 220 330 .04 .0001

CD191 B cells, % of lymphocytes 10.2 12 19.2 .5 .0021

Ratio ng BAFF/CD191 3 103 B cells 0.18 0.08 0.01 .08 .02

CD191CD21low B cells, % of CD191 B cells 25.5 12.35 7.8 .001 .0002

IgG, mg/dL 533 895 961 .0007 .0004

CD41 T cells, % of lymphocytes 23.6 21.6 23.4 .4 .9





corresponded to a sensitivity of 96% and a NPV of 94%. Thus,

analysis of CD191CD21low B cells in cGVHD is able to identify

almost all patients with incipient lung involvement when 80% of

them are still clinically asymptomatic. Patients with respiratory tract

infections had significantly fewer CD191CD21low B cells compared

with BOS, which is of major clinical importance, because clinical and

radiologic symptoms of pulmonary infection can mimic early BOS.

Significantly higher CD191CD21low B cells, BAFF, and

BAFF/CD191 B-cell ratios were observed in all cohorts with long-

lasting BOS compared with patients without cGVHD, confirming

the validity of our potential B-cell biomarkers for early diagnosis

of BOS. Patients with cutaneous cGVHD had significantly lower

CD191CD21low B cells, BAFF, and BAFF/CD191 B-cell ratio

compared with BOS patients. Thus, a high percentage of CD191

CD21low B cells can be regarded as a potential predictive marker

for the recent development of BOS in patients suffering from

cGVHD. Here, the cumulative incidence of BOS was 30%, whereas

Arai and colleagues3 recently reported a lower incidence of BOS

but 50% of lung involvement by cGVHD in 298 patients with

cGVHD prospectively assessed at study enrollment. Because the

Chronic GVHD Consortium of the U.S. did not provide any details

on the definitions used and had a substantially shorter follow-up

time, it must remain unanswered whether longer follow-up would

translate into a higher rate of BOS as seen in our study.

Historically, BOS was characterized as irreversible, progressive,

treatment-refractory obstruction. In our study, the 5-y OS for BOS

was 77% and NRM was 15%. Historically, survival of BOS

patients was poor7-10,13-15; however, previous publications in-

cluded patients with severe, long-lasting BOS diagnosed at later

time points using various diagnostic criteria that may not be

comparable with the NIH definition and thus hinder a valid

comparison. Arai and colleagues3 reported a 2-y OS of 86% and

62% for patients with moderate and severe NIH-defined cGVHD.

Almost 60% of our study population had severe cGVHD and

thus, our survival rates compare favorably with published results.

Significantly, survival of BOS patients diagnosed early reached

a level similar to that of patients without cGVHD. This is most

likely due to the fact that NRM was not significantly increased in

the BOS cohort, whereas risk for relapse was significantly increased

in patients without cGVHD as previously reported.6,37-39

In patients with BOS, CD191CD21lowCD38high immature transi-

tional B cells defined as previously reported16,17,40 were significantly

higher compared with patients without cGVHD. We also observed in

some patients with BOS a CD191CD21lowCD38lowCD10neg B-cell

population lacking CD27 coexpression which, so far, has only been

identified in HIV patients with high plasma viremia41 and patients

with common variable immunodeficiency (CVID).42 In CVID, these

CD21low B cells are believed to belong to a polyclonal population of

innate-like B cells of the non-memory pool that are enriched in PB,

synovial fluid of patients with rheumatoid arthritis, and BAL fluid in

the lungs of patients with CVID.42 CD21 represents an important

receptor for one of the potent immunoregulatory fragments of

complement component 3, ie, C-terminal C3d. CD21/complement

receptor 2 has several important functional roles within the

immune system.43 In mice, uncoupling of CD21 from the CD19/

CD81 complex led to enhanced apoptosis of germinal center B cells,

diminished secondary antibody titers and thus overall impairment

of humoral immunity.44 Therefore, CD21 not only delivers an

important signal for fine-tuning the threshold of B-cell activation

but also mediates an important survival signal for B cells within

germinal centers. Low to negative expression of CD21 on PB

B cells indicates that these cells are not in the position to exert such

functions. This represents the rationale for why we regard

CD21neg/low B cells as the group of B cells with altered co-receptor

capabilities and thus have subsumed the respective populations as

one entity.

Figure 4. Serial analyses of CD191CD21low B cells

from d 100 after allogeneic HCT until onset of and

during the course of BOS (B) compared with patients

without cGVHD (no cGVHD) (A). In both patient

cohorts, CD191CD21low B cells decreased 6 mo after

HCT, indicating B-cell reconstitution, because CD21low B

cells reportedly are the first B-cell population to be

exported from the bone marrow into the periphery and

decrease during B-cell maturation.33 Whereas in patients

without cGVHD (A) percentages of CD191CD21low B

cells further decreased until 11 mo after HCT, in patients

with newly diagnosed BOS (B) CD191CD21low B cells

increased again and remained elevated throughout the

course of BOS. Data are shown as box plots with

medians and ranges. ˚ indicates outliers.

Table 5. Confirmation of cellular and plasma biomarkers in patients with long-lasting BOS

Onset of BOS FEV1 < 55% Histological BO LFS 3 LSS 213 No cGVHD

Number of patients 46 23 23 9 11 41

BAFF, ng/mL 7.3* 8.0* 9.1* 8.0* 7.7* 3.5

CD191 B cells 3 106/L 146* 120* 117* 90* 129 317

CD191 B cells, % 10.2* 8.5* 8.7* 8.9* 8.5* 19.7

Ratio BAFF/CD191 0.18* 0.19* 0.18* 0.47* 0.21* 0.02

CD191CD21low, % 25.5* 27.8* 26.5* 22* 25.6* 6.6

CD41 T cells, % 23.62* 21.2* 21.8* 24.3* 23.2* 17.5

IgG, mg/dL 533* 580* 512* 584 449* 1172

*Indicates significant difference compared with patients without cGVHD.





Little is known regarding the pathogenesis of BOS after HCT.

Allorecognition of lung antigens is suspected, because new airflow

obstruction generally develops during or after recent withdrawal of

immunosuppression. Severe neutrophilic inflammation was ob-

served in the early phase of BOS, whereas prolonged immune

activation is known to induce a fibrotic process resulting in airway

obliteration.45 During airway wall destruction, bronchus-associated

lymphoid tissue is generated and B cells generate autoimmune

antibodies as reported against collagen V.46 Srinivasan et al.47

observed CD41 T-cell and donor-derived B2201 B-cell infiltra-

tions with alloantibody deposition in murine lungs with BO after

HCT. In our BOS cohort, BAFF levels and the BAFF/B-cell ratio

were significantly higher compared with patients without cGVHD.

BAFF promotes B-cell survival and proliferation and is known to

be produced by a variety of cells recruited during inflammation,

including activated airway epithelial cells leading to local accumu-

lation and activation of B cells.19,48

Although our results are promising, there are limitations to be

mentioned. This prospective cohort included only 46 patients with

BOS that were serially analyzed at defined time points after HCT

and the results were compared with those obtained in patients with

cutaneous cGVHD and patients without cGVHD matched for time

after HCT. A greater number of patients screened by PFTs,

radiology, and biomarker studies is thus warranted to confirm

our findings. Per design, this study was not an interventional one;

however, our findings suggest that a decrease in PFTs and elevation

of CD191CD21low B cells confirming the early diagnosis of BOS

identified patients with cGVHD who responded to early immuno-

suppressive treatment with corticosteroids. Additional prospective

studies will have to be performed to address the significance of

clinically asymptomatic lung disease such as NIH-defined mild

pulmonary involvement by cGVHD in terms of patient outcome

and long-term prognosis.

In conclusion, CD191CD21low B cells may serve as

a potential diagnostic biomarker for early onset of BOS, enable

the distinction of BOS from respiratory tract infection and other

organ manifestations of cGVHD, and could allow improvement

of patient outcome.

Acknowledgments

The authors thank Harald Fuehrer from the University of Vienna

for his assistance in statistical analyses.

This work was supported by European Commission grant 037703

STEMDIAGNOSTICS and a grant by Austrotransplant.

Authorship

Contribution: H.T.G. and W.F.P. designed the research study,

analyzed and interpreted the data, and coauthored the manuscript;

Z.K. and R.W. performed the clinical research and collected and

analyzed data; P.K. and M.M. performed clinical assessments of

cGVHD according to the NIH criteria; K.K. and V.P. performed

PFTs and clinical assessments; U.K. and A.R. performed flow

cytometric analyses; and L.P. evaluated HR-CT scans and G.D.

assessed transbronchial biopsies.

Conflict-of-interest disclosure: The authors declare no compet-

ing financial interests.

Correspondence: Hildegard T. Greinix, Medizinische Universi-

taet Wien, Klinik fuer Innere Medizin I, Knochenmarktransplanta-

tion, Waehringer Guertel 18-20, A-1090 Vienna, Austria; e-mail:

[email protected]; and Winfried F. Pickl,

Medizinische Universitaet Wien, Institut fuer Immunologie,

Borschkegasse 8a, A-1090 Vienna, Austria; e-mail: winfried.

[email protected].

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online January 9, 2013 originally publisheddoi:10.1182/blood-2012-06-435008

2013 121: 1886-1895

F. PicklPeter Kalhs, Margit Mitterbauer, Lothar Ponhold, Gerhard Dekan, Hildegard T. Greinix and Winfried Zoya Kuzmina, Katharina Krenn, Ventzislav Petkov, Ulrike Körmöczi, Roman Weigl, Arno Rottal, graft-versus-host disease with bronchiolitis obliterans syndrome

B cells and patients at risk for NIH-defined chroniclowCD21+CD19

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(2052 articles)Transplantation (5289 articles)Immunobiology

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cd19+cd21low b cells and patients at risk for nih-defined chronic graft-versus-host disease with...

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