Abstract

Manifestations of chronic graft-versus-host disease (cGVHD) can resemble those seen in immunodeficiency states and autoimmune disorders. Reports by us and others suggest an involvement of B cells in the pathogenesis of cGVHD. We investigated B-lymphocyte subpopulations in cGVHD cohorts defined by serum immunoglobulin G (IgG) levels to characterize novel biomarkers for impairment of humoral immunity after allogeneic hematopoietic stem cell transplantation. Seventy-six patients were enrolled a median of 46 months after hematopoietic stem cell transplantation. The hypogammaglobulinemia group had significantly diminished CD19+ B cells (165 vs 454 vs 417 × 106/L) with elevated CD19+CD21low immature (16.5%, 7.7%, and 9.1%) and CD19+CD21int-highCD38highIgMhigh transitional (10.5% vs 4.2% vs 6.3%) B-cell proportions compared with the normogammaglobulinemia and hypergammaglobulinemia groups. CD19+CD10CD27CD21high naive B cells were highly elevated in all patients with cGVHD. CD19+CD27+IgD+ non–class-switched (4 vs 12 vs 11 × 106/L) and class-switched (7 vs 35 vs 42 × 106/L) memory B cells were significantly lower in the hypogammaglobulinemia group compared with the others. Besides significantly higher B-cell activation factor/B-cell ratios, significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024). In conclusion, B-cell subpopulations can serve as novel cellular biomarkers for immunodeficiency and autoimmunity indicating different pathogenetic mechanisms of cGVHD and encouraging future prospective longitudinal studies.

Introduction

Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HCT) and is associated with increased nonrelapse mortality and prolonged immunodeficiency.1-3  Affected patients present unique clinical features, including lichenoid and sclerodermatous skin lesions, keratoconjunctivitis sicca, lichenoid mucositis, and bronchiolitis obliterans (BO) syndrome, which have recently been reclassified within the National Institutes of Health consensus development project.4  The clinical signs of cGVHD can resemble those seen in autoimmune disorders, such as systemic lupus erythematosus (SLE), Sjogren syndrome, scleroderma, and rheumatoid arthritis.5-7  Autoantibody formation has been frequently observed in these patients leading to autoimmune diseases, such as autoimmune-like thrombocytopenia, immune hemolytic anemia, and autoimmune thyroiditis.6,8-10 

The precise immunologic mechanisms leading to cGVHD currently still remain unclear.11,12  Because cGVHD only occurs in allogeneic HCT recipients and can be prevented by T-cell depletion from the donor graft, donor T cells responding to allogeneic antigens in the patient are of critical importance for the development of cGVHD. Of note, donor B-cell responses to recipient HY antigens have been significantly associated with the development of cGVHD in the setting of gender-mismatched HCT.13  Besides the presence of autoantibody abnormalities of B-cell function seen as distortion of B-cell homeostasis14  leading to uncontrolled overactivation of B cells recently emigrated from the bone marrow (BM) resulting from an excess of B-cell activating stimuli, such as B-cell activation factor (BAFF) characterize cGVHD.15  Lack of BAFF consumption by BAFF-R-expressing peripheral B-cell subpopulations due to B-lymphopenia is supposed to lead to an imbalance between the amount of bio-available BAFF and BAFF-R+ B cells. Consequently, large amounts of BAFF act on single B cells newly emigrated from the BM leading to the expansion of clones, which otherwise, under physiologic BAFF/B-cell ratios, would have been deleted or ignored.15 

Along those lines, we recently observed a significant increase of CD19+CD21 immature transitional B-cell numbers in patients with active cGVHD, which is reminiscent of findings in distinct autoimmune diseases, such as SLE and primary or acquired immunodeficiencies.14,16-19  Interestingly, the latter are regularly associated with hypogammaglobulinemia, which can also be observed in patients with cGVHD because the lymphoid system is known to be a target organ of cGVHD resulting in lymphoid hypocellularity, functional asplenia, and overall impaired immune reconstitution after HCT.20-22 

Here, we further explored the role of B-cell subpopulations in the pathogenesis of cGVHD by analyzing cohorts of cGVHD patients differing in their impairment of humoral immunity as reflected by changes in the generation of B cells and their antibody products as mirrored by serum immunoglobulin (Ig) levels. In addition, we correlated the size and distribution of B-cell subpopulations with autoimmune phenomena as detected by the presence of various autoantibodies in cGVHD patients' sera.

Methods

Trial conduct

From January 2009, all consecutive patients with cGVHD according to the National Institutes of Health consensus criteria,4  seen in the Outpatient Clinic of the Medical University of Vienna, Austria, were asked to participate in this study. Besides mild to severe cGVHD, inclusion criteria consisted of complete remission of malignant disease after HCT, lack of life-threatening infections, and written informed consent in accordance with the Declaration of Helsinki and the Institutional Review Board of the Medical University of Vienna. Besides clinical assessment for activity of cGVHD, peripheral blood samples were analyzed by flow cytometry, and serum Ig levels and the presence of autoantibodies were evaluated at the same time points.

Patients and therapeutic modalities

Seventy-six patients with cGVHD with a median age of 40 years (range, 23-59 years) were included into the study: 25 with hypergammaglobulinemia, 21 with hypogammaglobulinemia, and 30 with normogammaglobulinemia. Patients were assigned to a subgroup according to values of at least 2 of the 3 major immunoglobulin isotypes (IgM, IgG, and IgA). Transplantation and patient characteristics are shown in Tables 1 and 2, including mean serum levels of IgG, IgM, and IgA for all 3 subgroups, respectively.

Table 1

Characteristics of all transplant recipients and patient subgroups

All, n (%)Hypergammaglobulinemia, n (%)Hypogammaglobulinemia, n (%)Normogammaglobulinemia, n (%)
No. of patients 76 25 21 30 
Median age, y (range) 40 (23-59) 39 (23-54) 40 (23-51) 42 (29-59) 
Sex     
    Male 41 16 12 13 
    Female 35 17 
Diagnosis     
    Acute leukemia 36 13 10 13 
    Chronic myeloid leukemia 14 
    Malignant lymphoma 14 
    Other* 12 
Disease status at transplantation     
    Standard risk 43 14 20 
    High risk 33 11 12 10 
Conditioning regimen     
    Myeloablative 45 13 12 20 
    Reduced intensity 31 12 10 
Stem cell donors     
    Related 24 
    Unrelated 52 16 15 21 
    HLA-identical 66 21 19 26 
    HLA-mismatched 10 
Stem cell source     
    Bone marrow 
    Peripheral blood 70 23 19 28 
Median no. CD34+ cells × 106/kg transplanted (range) 5.6 (0.9-10.4) 6.5 (2.9-10.4) 5.9 (1.7-8.2) 5.6 (0.9-7.6) 
Posttransplantation immunosuppressive prophylaxis     
    Cyclosporine only 
    Cyclosporine-methotrexate 41 14 14 13 
    Cyclosporine-mycophenolate mofetil 31 11 14 
Acute GVHD     
    Grade 0 32 17 
    Grade I or II 26 10 
    Grade III or IV 18 
All, n (%)Hypergammaglobulinemia, n (%)Hypogammaglobulinemia, n (%)Normogammaglobulinemia, n (%)
No. of patients 76 25 21 30 
Median age, y (range) 40 (23-59) 39 (23-54) 40 (23-51) 42 (29-59) 
Sex     
    Male 41 16 12 13 
    Female 35 17 
Diagnosis     
    Acute leukemia 36 13 10 13 
    Chronic myeloid leukemia 14 
    Malignant lymphoma 14 
    Other* 12 
Disease status at transplantation     
    Standard risk 43 14 20 
    High risk 33 11 12 10 
Conditioning regimen     
    Myeloablative 45 13 12 20 
    Reduced intensity 31 12 10 
Stem cell donors     
    Related 24 
    Unrelated 52 16 15 21 
    HLA-identical 66 21 19 26 
    HLA-mismatched 10 
Stem cell source     
    Bone marrow 
    Peripheral blood 70 23 19 28 
Median no. CD34+ cells × 106/kg transplanted (range) 5.6 (0.9-10.4) 6.5 (2.9-10.4) 5.9 (1.7-8.2) 5.6 (0.9-7.6) 
Posttransplantation immunosuppressive prophylaxis     
    Cyclosporine only 
    Cyclosporine-methotrexate 41 14 14 13 
    Cyclosporine-mycophenolate mofetil 31 11 14 
Acute GVHD     
    Grade 0 32 17 
    Grade I or II 26 10 
    Grade III or IV 18 

HLA indicates human leukocyte antigen.

*

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 and malignant lymphoma.

Table 2

Description of cGVHD

All, n (%)Hypergammaglobulinemia, n (%)Hypogammaglobulinemia, n (%)Normogammaglobulinemia, n (%)
No. of patients 76 25 21 30 
Organ involvement at study entry 
    Skin 47 (62) 16 (64) 15 (71) 16 (53) 
    Deep cutaneous sclerosis 17 (22) 11 (44) 6 (29) 
    Eyes 26 (34) 11 (44) 13 (62) 2 (6) 
    Oral mucosa 43 (57) 17 (68) 12 (57) 14 (47) 
    Liver 24 (32) 13 (52) 7 (33) 4 (13) 
    Lungs 21 (28) 10 (40) 10 (48) 1 (3) 
    GI 6 (8) 2 (8) 3 (14) 1 (3) 
    Genital* 7 (20) 2 (22) 3 (33) 2 (12) 
    Joints 11 (14) 5 (20) 3 (14) 3 (10) 
Onset type 
    De novo 33 (44) 11 (44) 6 (28) 16 (53) 
    Quiescent 26 (34) 9 (36) 9 (43) 8 (27) 
    Progressive 17 (22) 5 (20) 6 (28.5) 6 (20) 
NIH severity score 
    Mild 7 (9) 3 (12) 1 (5) 3 (10) 
    Moderate 19 (25) 5 (20) 2 (9) 12 (40) 
    Severe 50 (66) 17 (68) 18 (86) 15 (50) 
No. of organs involved 
    ≤ 2 27 (36) 11 (44) 4 (19) 12 (40) 
    > 2 49 (64) 14 (56) 17 (81) 18 (60) 
Median duration of cGVHD 
    mo 42 29 45 63 
    (range) (3-136) (3-92) (3-136) (26-110) 
Immunosuppressive therapy 
    CSA/FK with or without MMF with or without steroids 18 (24) 3 (12) 7 (33) 8 (27) 
    ECP with or without steroids 12 (16) 7 (28) 5 (24) 
    Sirolimus with or without steroids 12 (16) 5 (20) 7 (33) 
    Steroids alone 21 (28) 9 (36) 2 (10) 10 (33) 
    No systemic therapy 13 (17) 1 (4) 12 (40) 
No. of IS therapies before study entry     
    1 21 (28) 7 (28) 2 (10) 12 (40) 
    2 25 (33) 7 (28) 4 (19) 14 (47) 
    ≥ 3 30 (39) 11 (44) 15 (71) 4 (13) 
Mean IgG, mg/dL 1441 3040 226 960 
    (range) (100-3950) (2320-3950) (100-395) (750-1300) 
Mean IgM, mg/dL 87 127 52 78 
    (range) (12-507) (36-507) (12-84) (16-133) 
Mean IgA, mg/dL 104 141 80 108 
    (range) (6-735) (17-735) (6-155) (6-273) 
No. of patients with autoantibodies present 24 (32) 17§ (68) 5§ (24) 2 (7) 
No. of patients with autoantibodies absent 52 (68) 8 (32) 16 (76) 28 (93) 
No. of patients with ANA present 24 (32) 17 (68) 5 (24) 2 (7) 
Mean ANA, IU/mL 764 926 762 320 
    (range) (70-1280) (70-1280) (320-1280) (160-320) 
    > 1250 10 
    > 640 ≤ 1250 
    ≤ 640 
No. of patients with anti-dsDNA present 7 (9) 5 (20) 1 (5) 1 (3) 
Mean anti-dsDNA, IU/mL 23.5 26 13 13 
    (range) (13-66) (13-66) NA NA 
All, n (%)Hypergammaglobulinemia, n (%)Hypogammaglobulinemia, n (%)Normogammaglobulinemia, n (%)
No. of patients 76 25 21 30 
Organ involvement at study entry 
    Skin 47 (62) 16 (64) 15 (71) 16 (53) 
    Deep cutaneous sclerosis 17 (22) 11 (44) 6 (29) 
    Eyes 26 (34) 11 (44) 13 (62) 2 (6) 
    Oral mucosa 43 (57) 17 (68) 12 (57) 14 (47) 
    Liver 24 (32) 13 (52) 7 (33) 4 (13) 
    Lungs 21 (28) 10 (40) 10 (48) 1 (3) 
    GI 6 (8) 2 (8) 3 (14) 1 (3) 
    Genital* 7 (20) 2 (22) 3 (33) 2 (12) 
    Joints 11 (14) 5 (20) 3 (14) 3 (10) 
Onset type 
    De novo 33 (44) 11 (44) 6 (28) 16 (53) 
    Quiescent 26 (34) 9 (36) 9 (43) 8 (27) 
    Progressive 17 (22) 5 (20) 6 (28.5) 6 (20) 
NIH severity score 
    Mild 7 (9) 3 (12) 1 (5) 3 (10) 
    Moderate 19 (25) 5 (20) 2 (9) 12 (40) 
    Severe 50 (66) 17 (68) 18 (86) 15 (50) 
No. of organs involved 
    ≤ 2 27 (36) 11 (44) 4 (19) 12 (40) 
    > 2 49 (64) 14 (56) 17 (81) 18 (60) 
Median duration of cGVHD 
    mo 42 29 45 63 
    (range) (3-136) (3-92) (3-136) (26-110) 
Immunosuppressive therapy 
    CSA/FK with or without MMF with or without steroids 18 (24) 3 (12) 7 (33) 8 (27) 
    ECP with or without steroids 12 (16) 7 (28) 5 (24) 
    Sirolimus with or without steroids 12 (16) 5 (20) 7 (33) 
    Steroids alone 21 (28) 9 (36) 2 (10) 10 (33) 
    No systemic therapy 13 (17) 1 (4) 12 (40) 
No. of IS therapies before study entry     
    1 21 (28) 7 (28) 2 (10) 12 (40) 
    2 25 (33) 7 (28) 4 (19) 14 (47) 
    ≥ 3 30 (39) 11 (44) 15 (71) 4 (13) 
Mean IgG, mg/dL 1441 3040 226 960 
    (range) (100-3950) (2320-3950) (100-395) (750-1300) 
Mean IgM, mg/dL 87 127 52 78 
    (range) (12-507) (36-507) (12-84) (16-133) 
Mean IgA, mg/dL 104 141 80 108 
    (range) (6-735) (17-735) (6-155) (6-273) 
No. of patients with autoantibodies present 24 (32) 17§ (68) 5§ (24) 2 (7) 
No. of patients with autoantibodies absent 52 (68) 8 (32) 16 (76) 28 (93) 
No. of patients with ANA present 24 (32) 17 (68) 5 (24) 2 (7) 
Mean ANA, IU/mL 764 926 762 320 
    (range) (70-1280) (70-1280) (320-1280) (160-320) 
    > 1250 10 
    > 640 ≤ 1250 
    ≤ 640 
No. of patients with anti-dsDNA present 7 (9) 5 (20) 1 (5) 1 (3) 
Mean anti-dsDNA, IU/mL 23.5 26 13 13 
    (range) (13-66) (13-66) NA NA 

GI indicates gastrointestinal; NIH, National Institutes of Health; CSA, cyclosporine A; FK, tacrolimus; ECP, extracorporeal photopheresis; MMF, mycophenolate mofetil; IS, immunosuppressive; ANA, antinuclear antibody; anti-dsDNA, antibody against double-stranded DNA; and NA, not applicable.

*

Only female patients.

P = .04.

P < .0001.

§

P = .024.

Diagnosis of cGVHD and its severity were performed according to the National Institutes of Health consensus criteria.4  The most frequent organ manifestations of cGVHD at study entry were skin (62%) and oral mucosa (57%). Forty-nine patients (64%) had more than 2 organs involved, and 50 patients (66%) had severe cGVHD. No significant differences in organ manifestations of cGVHD were observed between the subgroups, but patients with hypogammaglobulinemia had significantly (P = .04) more severe cGVHD as shown in Table 2. In the patient cohort with hypergammaglobulinemia, the median duration of cGVHD was significantly shorter (29 vs 63 months, P < .0001) compared with the cohort with normogammaglobulinemia. Furthermore, significantly (P = .024) more patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup.

All patients received anti-infective prophylaxis as described.23  Immunosuppressive therapies at study entry are shown in Table 2.

Isolation of blood cells, immunophenotyping, and flow cytometry

Optimal concentrations of directly conjugated monoclonal antibodies (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) were added to 50 μL of patients' and healthy volunteers' whole blood, and incubated for 20 minutes at room temperature. ADG-lysis solution (An der Grub) was used to remove red blood cells according to the manufacturer's recommendations followed by acquisition of 5 × 103 cells in the lymphogate for leukocyte subpopulations and 4 to 8 × 103 CD19+ B cells for B-cell subset analysis, as described14  and shown in Figure 1. Naive CD4+ T cells were defined as CD4+CD45RA+, naive CD8+ T cells as CD8+CD45RA+, memory CD4+ T cells as CD4+CD45RA, and memory CD8+ T cells as CD8+CD45RA cells, respectively.

Figure 1

Flow cytometric gating algorithm demonstrating circulating B-cell subpopulations in patients with cGVHD. Whole blood was analyzed by multicolor flow cytometry, and CD19+ B cells were further stained in various combinations for CD21, CD27, CD38, CD10, IgM, and IgD. Immature, transitional, naive, non–class-switched and class-switched memory B cells were enumerated, which were different between healthy donors and patients with cGVHD.

Figure 1

Flow cytometric gating algorithm demonstrating circulating B-cell subpopulations in patients with cGVHD. Whole blood was analyzed by multicolor flow cytometry, and CD19+ B cells were further stained in various combinations for CD21, CD27, CD38, CD10, IgM, and IgD. Immature, transitional, naive, non–class-switched and class-switched memory B cells were enumerated, which were different between healthy donors and patients with cGVHD.

Assessment of peripheral blood immunoglobulin levels and autoantibodies

Serum levels of IgG, IgM, and IgA were quantified by nephelemetry (BNII, Dade Behring). The normal values for IgG were 700 to 1600 mg/dL, for IgA 70 to 400 mg/dL, and for IgM 40 to 230 mg/dL, respectively. In all patients with elevation of immunoglobulin levels, standard serum protein electrophoresis was performed to exclude oligoclonal B-cell reconstitution. The following autoantibodies were investigated by enzyme immunoassays according to the manufacturer's protocol (Siemens): antinuclear antibody, antibody against double-stranded DNA, antibody against histones, extractable nuclear antigen antibody, lupus erythematosus antibody, scleroderma antibody, antibody against SM antigen, antibody against ribonucleoprotein, autoimmune myositis antibody, antibody against topoisomerase, antibody against thyroid hormone, antibody against thyroid-stimulating hormone receptor, and liver-kidney antibody. For correlation of autoantibody levels and cGVHD characteristics, the most frequently observed autoantibodies, namely, antinuclear antibody (ANA) levels were divided into 3 groups consisting of more than 1250 IU/mL, 1250 to 640 IU/mL, and less than 640 IU/mL, respectively.

Assessment of BAFF by enzyme-linked immunosorbent assay

Patients' plasma samples were examined for sBAFF by enzyme-linked immunosorbent assay (R&D Systems) according to the manufacturer's instructions.

Statistical analysis

In the figures, data are shown as box plots. The box represents the middle half of the distribution of the data points stretching from the 25th percentile (“lower hinge”) to the 75th percentile (“upper hinge”). The line across the box represents the mean. The lengths of the lines above and below the box (“inner fences”) are defined by the maximum and minimum data point values that lie within 1.5 times the spread of the box. Outlying origins that do not fall within the inner fences are shown as open circles. Outlying points typically correspond to origins not identified by the respective study.

Fisher exact test was used to examine the significance of the association between 2 variables. Statistical pair-wise comparisons of B-cell subsets and BAFF levels with each Ig subgroup were made using unpaired Student t test. A multivariate analysis for factors impacting on B-cell subpopulations, BAFF levels, and serum Ig levels was performed. For univariate analysis on the association of different B-cell subpopulations with cGVHD characteristics, variables were selected according to the most frequent organ manifestations of cGVHD (bronchiolitis obliterans, skin, eye, and oral mucosa), severity of cGVHD (mild/moderate vs severe) and number of organs involved (< 2 vs > 2), and presentations known to be associated with humoral immunity (scleroderma, autoimmune hepatitis, and the presence or absence of autoantibodies), respectively. Covariates with a P value less than .05 were entered into the multivariate analysis. As a second step, multiple logistic regression analyses were calculated for each of the main organ manifestations.

The influence of immunosuppressive therapies on B-cell subpopulations was addressed by univariate analysis of variance using different therapeutic groups (calcineurin inhibitor based, sirolimus based, extracorporeal photopheresis based, steroids alone, no immunosuppressive therapy). All pair-wise comparisons were corrected for Bonferroni. Those B-cell subpopulations that showed a significant correlation in the univariate analysis with an immunosuppressive regimen were further analyzed in a multivariate analysis using as variables therapeutic groups and severity of cGVHD.

The data were calculated using SPSS Version 16.0 (IBM).

Results

We included 76 patients after a median duration of 42 months (range, 3-136 months) of cGVHD into the study (Tables 12). All were in remission of their original hematologic disease and had complete multilineage donor chimerism as determined by standard variable number of tandem repeat analysis. No significant differences in absolute CD3+, CD4+, CD8+, and NK cell numbers were observed between cGVHD subgroups defined according to serum immunoglobulin levels (not shown). In addition, CD4+CD45RA+ naive CD4, CD8+CD45RA+ naive CD8, CD4+CD45RA memory CD4, and CD8+CD45RA memory CD8 T cells were not significantly different between the cGVHD cohorts.

cGVHD patients with hypogammaglobulinemia have a significant B-cell deficiency and an increased BAFF/B-cell ratio

We and others observed a significant distortion of B-cell subpopulations and high plasma BAFF levels in patients with cGVHD.14,15  Here, we performed further multiparameter flow cytometric analyses of B-cell subsets in patient cohorts with cGVHD distinguished by their serum Ig levels (Figure 1). At a median of 46 months (range, 3-171 months) after HCT, a median number of 350 × 106/L CD19+ B cells was observed in the overall study population. Although CD19+ B-cell numbers were comparable in cGVHD patients with hypergammaglobulinemia and normogammaglobulinemia (417 vs 454 × 106/L), the cGVHD cohort with hypogammaglobulinemia had significantly decreased CD19+ B-cell numbers compared with the normogammaglobulinemia (165 vs 454 × 106/L, P < .001) and the hypergammaglobulinemia cohorts (165 vs 417 × 106/L, P < .01), as shown in Figure 2A.

Figure 2

CD19+ B cells and BAFF/B cell ratio in cGVHD patients with dysgammaglobulinemia. (A) cGVHD patients with hypogammaglobulinemia have a significant CD19+ B-cell deficiency. “Hyper Ig” represents cGVHD patients with IgG more than 1600 mg/dL, “Hypo Ig” with IgG less than 700 mg/dL, and “Normal Ig” with IgG 700 to 1600 mg/dL. Lines indicate the range of CD19+ B cells in healthy persons. Data are shown as box plots. (B) Significant elevation of BAFF/B-cell ratios (nanograms per 1 × 103 B cells) in patients with cGVHD and dysgammaglobulinemia. Data are shown as box plots. ○ represents outliers.

Figure 2

CD19+ B cells and BAFF/B cell ratio in cGVHD patients with dysgammaglobulinemia. (A) cGVHD patients with hypogammaglobulinemia have a significant CD19+ B-cell deficiency. “Hyper Ig” represents cGVHD patients with IgG more than 1600 mg/dL, “Hypo Ig” with IgG less than 700 mg/dL, and “Normal Ig” with IgG 700 to 1600 mg/dL. Lines indicate the range of CD19+ B cells in healthy persons. Data are shown as box plots. (B) Significant elevation of BAFF/B-cell ratios (nanograms per 1 × 103 B cells) in patients with cGVHD and dysgammaglobulinemia. Data are shown as box plots. ○ represents outliers.

Moreover, cGVHD patients with hypergammaglobulinemia and hypogammaglobulinemia had significantly higher BAFF/B-cell ratios compared with the normogammaglobulinemia cohort (0.01 and 0.03 vs 0.005 ng BAFF/103 CD19+ B cells, P < .05; Figure 2B).

cGVHD patients with hypogammaglobulinemia have a most pronounced distortion of B-cell homeostasis

Next, we addressed the distribution of various B-cell subpopulations of importance for maintenance of healthy immune homeostasis. CD19+CD21low immature B cells were elevated in proportion to other more mature B-cell subpopulations in all patients with cGVHD compared with healthy controls, as reported14  and as shown in Figure 3A. In addition, patients with hypogammaglobulinemia had significantly higher proportions of CD19+CD21low immature B cells compared with cGVHD patients with hypergammaglobulinemia (16.5% vs 9.1%, P < .01) or normogammaglobulinemia (16.5% vs 7.7%, P = .002). CD19+CD21int-highCD38highIgMhigh transitional B cells were relatively increased in all patients with cGVHD compared with healthy controls14  and were significantly higher in patients with hypogammaglobulinemia compared with the normogammaglobulinemia cohorts (10.5% vs 4.2%, P = .03) as shown in Figure 3B. No significant difference in proportion of CD19+CD21int-highCD38highIgMhigh transitional B cells between the hypogammaglobulinemia and hypergammaglobulinemia subgroups (10.5% vs 6.3%, P = .1) was observed. CD19+CD10CD27CD21high naive B cells were highly elevated in all patients with cGVHD compared with healthy controls (85% vs 50%, not shown). When looking at absolute cell numbers, significantly lower CD19+CD10CD27CD21high naive B cells were observed in cGVHD patients with hypogammaglobulinemia compared with the hypergammaglobulinemia (125 vs 342 × 106/L, P = .004) and normogammaglobulinemia cohorts (125 vs 346 × 106/L, P = .02), as shown in Figure 3C.

Figure 3

Most pronounced distortion of B-cell homeostasis in cGVHD patients with hypogammaglobulinemia. (A) Percentages of CD19+CD21low immature B cells are significantly elevated in cGVHD patients and mainly in the cohort with hypogammaglobulinemia. Lines indicate the range of CD19+CD21low immature B cells in healthy persons (2%–7%). Data are shown as box plots. (B) CD19+CD21int-highCD38highIgMhigh transitional B cells are significantly higher in cGVHD patients with hypogammaglobulinemia compared with the normogammaglobulinemia cohort. The line indicates the normal upper range of these cells in healthy persons. Data are shown as box plots. ○ represents outliers. (C) Significantly lower absolute numbers of CD19+CD10CD27CD21high naive B cells in cGVHD patients with hypogammaglobulinemia compared with the hypergammaglobulinemia and normogammaglobulinemia cohort. We observed a mean percentage of 85 in cGVHD patients compared with 50% of CD19+CD10CD27CD21high naive B cells in healthy persons. Data are shown as box plots. (D) CD19+CD27+IgD+ non–class-switched memory B cells are significantly lower in cGVHD patients with hypogammaglobulinemia compared with the hypergammaglobulinemia and normogammaglobulinemia cohort. Data are shown as box plots.

Figure 3

Most pronounced distortion of B-cell homeostasis in cGVHD patients with hypogammaglobulinemia. (A) Percentages of CD19+CD21low immature B cells are significantly elevated in cGVHD patients and mainly in the cohort with hypogammaglobulinemia. Lines indicate the range of CD19+CD21low immature B cells in healthy persons (2%–7%). Data are shown as box plots. (B) CD19+CD21int-highCD38highIgMhigh transitional B cells are significantly higher in cGVHD patients with hypogammaglobulinemia compared with the normogammaglobulinemia cohort. The line indicates the normal upper range of these cells in healthy persons. Data are shown as box plots. ○ represents outliers. (C) Significantly lower absolute numbers of CD19+CD10CD27CD21high naive B cells in cGVHD patients with hypogammaglobulinemia compared with the hypergammaglobulinemia and normogammaglobulinemia cohort. We observed a mean percentage of 85 in cGVHD patients compared with 50% of CD19+CD10CD27CD21high naive B cells in healthy persons. Data are shown as box plots. (D) CD19+CD27+IgD+ non–class-switched memory B cells are significantly lower in cGVHD patients with hypogammaglobulinemia compared with the hypergammaglobulinemia and normogammaglobulinemia cohort. Data are shown as box plots.

As previously reported,14  both CD19+CD27+IgD+ non–class-switched and CD19+CD27+IgD class-switched memory B cells were profoundly decreased in absolute cell numbers and proportions in all patients with cGVHD. For valid correlation of memory B-cell numbers with serum immunoglobulin levels, absolute cell numbers were assessed revealing significantly lower CD19+CD27+IgD+ non–class-switched memory B cells in patients with hypogammaglobulinemia compared with the hypergammaglobulinemia (4 vs 11 × 106/L, P = .03) and the normogammaglobulinemia cohorts (4 vs 12 × 106/L, P < .001), as shown in Figure 3D. In addition, CD19+CD27+IgD class-switched memory B cells were significantly diminished in the hypogammaglobulinemia subgroup compared with cGVHD patients with normogammaglobulinemia (7 vs 35 × 106/L, P < .001, not shown), whereas no significant differences between the hypergammaglobulinemia and normogammaglobulinemia subgroups (42 vs 35 × 106/L, P = .78) and the hypergammaglobulinemia and hypogammaglobulinemia subgroups (42 vs 7 × 106/L, P = .2) were seen.

In addition, no significant differences in the numbers of circulating plasmablasts were observed between the cGVHD cohorts.

cGVHD patients with hypergammaglobulinemia have a significantly increased BAFF/B-cell ratio and serum autoantibodies

Besides significantly higher BAFF/B-cell ratios compared with patients with normogammaglobulinemia (P = .03), as shown in Figure 2B, significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024; Table 2). Although in all patients with detectable autoantibodies ANA were present, only 7 of 24 (29%) had anti-dsDNA autoantibodies with a mean level of 23.5 IU/mL (range, 13-66 IU/mL; Table 2), respectively. In addition, in the hypergammaglobulinemia subgroup, 2 patients had anticentromer B and one patient liver-kidney autoantibodies present; whereas in the hypogammaglobulinemia subgroup, one patient had antithyroperoxidase-A autoantibodies, respectively.

In multivariate analysis, the presence of ANAs was significantly associated with scleroderma (P = .032), autoimmune hepatitis (P < .001), and more than 2 organs involved by cGVHD (P = .018). In addition, patients with ANAs had significantly higher CD19+CD21low immature B cells (P = .009) compared with cGVHD patients without autoantibodies.

B-cell subpopulations in cGVHD patients with different organ involvements

Next, we analyzed B-cell subpopulations according to main organ involvement by cGVHD, as shown in Table 3. Patients with cGVHD of the eyes (n = 26) had the lowest CD19+ B-cell and the highest CD19+CD21int-highCD38highIgMhigh transitional B-cell numbers. In patients with lung manifestations of cGVHD (n = 21) presented as BO, the overall number of CD19+ was significantly reduced (mean 258 vs 428 × 106/L, P = .04), CD19+CD21low immature B-cell proportions were most pronouncedly elevated (mean 16.1 vs 8.04%, P = .002), CD19+CD10CD27CD21high naive B-cell proportions lowest (mean 77 vs 85%, P = .009), CD19+CD27+IgD+ non–class-switched memory B cells diminished (mean 4 vs 10 × 106/L, P = .02), and BAFF levels were increased (mean 2.3 vs 1.1 ng/mL, P = .04) compared with other organ manifestations.

Table 3

B-cell subpopulations and clinical characteristics

ParameterScleroderma
Bronchiolitis obliteraus
Mild/moderate vs severe
≤ 2 vs > 2 organs involved
YesNoPYesNoPMild/moderateSevereP≤ 2> 2P
CD19+ B cells × 106/L 302 453 .06 258 428 .041* 420 207 .07 405 227 .019* 
Immature B cells absolute × 106/L 26 28 .74 25 28 .57 26 26 .7 31 23 .8 
    % 14.1 7.7 .02* 16.1 8.04 .002* 9.2 14.5 .54 9.2 5.7 .07 
Transitional B cells absolute × 106/L 26 23 .78 11 23 .25 19 18 .6 22 15 .3 
    % 7.2 4.1 .18 6.2 .38 7.2 .48 6.1 3.6 .15 
Naive B cells absolute × 106/L 270 380 .2 200 360 .05 360 170 .07 340 170 .03* 
    % 84 84 .9 77 85 .0089* 86 79 .012* 85 83 .58 
Class-switched memory B cells absolute × 106/L 10 40 .005* 25 30 .6 26 11 .44 30 10 .038* 
    % 6.1 .28 7.6 .41 6.3 7.8 .03* 6.3 7.9 .55 
Non–class-switched memory B cells absolute × 106/L 10 .037* 10 .017* 10 .0096* 10 .022* 
    % 3.01 3.15 .85 2.7 2.9 .83 3.2 2.4 .17 3.1 2.3 .28 
BAFF, ng/mL 1.7 0.76 .0001* 2.3 1.1 .04* 1.78 1.9 .39 1.6 0.8 .18 
IgG, mg/dL 1955 1124 .001* 1168 1113 .82 1396 1108 .65 1324 1106 .17 
Ratio BAFF/immature B cells 0.09 0.05 .01* 0.12 0.07 .09 0.01 0.02 .9 0.12 0.06 .4 
Ratio BAFF/memory B cells 0.17 0.04 .02* 0.24 0.12 .4 0.12 0.15 .86 0.18 0.04 .33 
Ratio BAFF/CD19+ B cells 0.01 0.004 .08 0.01 0.0063 .03* 0.21 0.23 .4 0.01 0.002 .5 
ParameterScleroderma
Bronchiolitis obliteraus
Mild/moderate vs severe
≤ 2 vs > 2 organs involved
YesNoPYesNoPMild/moderateSevereP≤ 2> 2P
CD19+ B cells × 106/L 302 453 .06 258 428 .041* 420 207 .07 405 227 .019* 
Immature B cells absolute × 106/L 26 28 .74 25 28 .57 26 26 .7 31 23 .8 
    % 14.1 7.7 .02* 16.1 8.04 .002* 9.2 14.5 .54 9.2 5.7 .07 
Transitional B cells absolute × 106/L 26 23 .78 11 23 .25 19 18 .6 22 15 .3 
    % 7.2 4.1 .18 6.2 .38 7.2 .48 6.1 3.6 .15 
Naive B cells absolute × 106/L 270 380 .2 200 360 .05 360 170 .07 340 170 .03* 
    % 84 84 .9 77 85 .0089* 86 79 .012* 85 83 .58 
Class-switched memory B cells absolute × 106/L 10 40 .005* 25 30 .6 26 11 .44 30 10 .038* 
    % 6.1 .28 7.6 .41 6.3 7.8 .03* 6.3 7.9 .55 
Non–class-switched memory B cells absolute × 106/L 10 .037* 10 .017* 10 .0096* 10 .022* 
    % 3.01 3.15 .85 2.7 2.9 .83 3.2 2.4 .17 3.1 2.3 .28 
BAFF, ng/mL 1.7 0.76 .0001* 2.3 1.1 .04* 1.78 1.9 .39 1.6 0.8 .18 
IgG, mg/dL 1955 1124 .001* 1168 1113 .82 1396 1108 .65 1324 1106 .17 
Ratio BAFF/immature B cells 0.09 0.05 .01* 0.12 0.07 .09 0.01 0.02 .9 0.12 0.06 .4 
Ratio BAFF/memory B cells 0.17 0.04 .02* 0.24 0.12 .4 0.12 0.15 .86 0.18 0.04 .33 
Ratio BAFF/CD19+ B cells 0.01 0.004 .08 0.01 0.0063 .03* 0.21 0.23 .4 0.01 0.002 .5 
*

Significant P value.

Among 47 patients with cutaneous cGVHD, 17 had sclerodermatous manifestations together with significantly higher CD19+CD21low immature B-cell proportions (mean 14.1% vs 7.7%, P = .02), lower CD19+CD27+IgD+ non–class-switched (mean 7 vs 10 × 106/L, P = .04) and CD19+CD27+IgD class-switched memory B-cell numbers (mean 10 vs 40 × 106/L, P = .005), and higher BAFF levels (mean 1.7 vs 0.76 ng/mL, P = .0001) compared with cGVHD patients without scleroderma.

Patients with more than 2 organs involved by cGVHD had lower CD19+ B-cell numbers (mean 227 vs 405 × 106/L, P = .02), lower CD19+CD10CD27CD21high naive B cells (170 vs 340 × 106/L, P = .03), lower CD19+CD27+IgD+ non–class-switched (mean 5 vs 10 × 106/L, P = .02), and CD19+CD27+IgD class-switched memory B-cell numbers (mean 10 vs 30 × 106/L, P = .04) compared with cGVHD patients with 2 or less organs affected.

Patients with severe cGVHD had significantly lower CD19+CD10CD27CD21high naive B-cell proportions (mean 79 vs 86%, P = .01) and lower CD19+CD27+IgD+ non–class-switched memory B cells (4 vs 10 × 106/L, P = .001) compared with ones with mild and moderate cGVHD.

Moreover, in multiple logistic regression analyses performed for the main organ manifestations of cGVHD, scleroderma was found to be significantly associated with hypergammaglobulinemia (P = .02).

B-cell subpopulations in cGVHD patients with different intensity of systemic immunosuppressive therapies

As shown in Table 2, at study entry 18 patients were on immunosuppression with calcineurin inhibitors, 12 on extracorporeal photopheresis, 12 on sirolimus, and 21 on steroids alone, whereas 13 patients did not require systemic immunosuppressive therapy. We assessed distribution of B-cell subpopulations, BAFF, and immunoglobulin levels of the various therapeutic cohorts.

In univariate analysis, no significant differences in B-cell subpopulations, BAFF, and immunoglobulin levels were observed both between the various therapeutic groups as well as cGVHD patients given first-line, second-line, or third-line immunosuppressive therapies.

Discussion

During the last years, an increasingly important role of B cells in the pathogenesis of cGVHD has been reported.11,14,15,24-27  We recently observed a significant distortion of B-cell homeostasis seen in significant elevation of CD19+CD21low immature B cells and deficiency of CD19+CD27+ memory B cells in patients with active cGVHD.14  Because other hallmarks of affection of the patients' immune system by cGVHD are hypogammaglobulinemia20,28,29  and functional hyposplenism, which can occur in up to 15% of HCT patients21,30  on the one hand and hypergammaglobulinemia31,32  and presence of autoantibodies at onset and related to activity of cGVHD on the other hand,20,33,34  we analyzed different cGVHD patient cohorts defined by serum Ig levels as surrogate markers for B-cell function. We observed in cGVHD patients with hypogammaglobulinemia a significant CD19+ B-cell deficiency with significantly higher CD19+CD21low immature B-cell proportions, significantly higher CD19+CD21int-highCD38highIgMhigh transitional B-cell proportions, significantly lower CD19+CD10CD27CD21high naive B cells and significantly lower CD19+CD27+IgD+ non–class-switched and CD19+CD27+IgD class-switched memory B cells compared with cGVHD patients with hypergammaglobulinemia or normogammaglobulinemia. Recently, D'Orsogna et al observed a significant association of a history of GVHD with lower serum levels of IgG and lower circulating memory B-cell numbers.35  Glas et al reported 9 patients with cGVHD with low circulating memory B-cell numbers and a B-cell autonomous somatic mutation deficit one year after allogeneic HCT, suggesting a B-cell intrinsic inability to be driven to accumulate somatic mutations after HCT.36  Furthermore, damage of germinal centers of peripheral secondary lymphoid organs by cGVHD, as seen in hyposplenism, could lead to impaired B-cell differentiation and class switch recombination resulting in lack of memory B cells in patients with cGVHD that is even more severe in cohorts with hypogammaglobulinemia. However, the observed disturbance of B-cell homeostasis in cGVHD patients where the B-cell compartment was primarily composed of immature and transitional B cells that do not participate in germinal center reactions and do not acquire somatic mutations could also suggest that certain B-cell subpopulations are directly involved in the pathogenesis of cGVHD. Deficiency of memory B cells, and predominance of transitional B cells in the peripheral blood B-cell compartment seem to be a common feature of immunodeficiency states because similar findings have been reported in patients with X-linked lymphoproliferative disease,19  common variable immunodeficiency16,37  and HIV infection.18  All these clinical conditions are characterized by hypogammaglobulinemia and susceptibility for severe infections resulting from an impaired ability to mount efficient humoral immune responses. These disturbances of B-cell homeostasis could contribute to the patients' status of immunodeficiency because transitional B cells are known to produce less Ig than mature B cells.19  Thus, enumerating B-cell subpopulations may be a novel tool for assessing the immunocompetence of cGVHD patients. Recently, Corre et al reported a significant deficiency of memory B cells 12 and 24 months after HCT together with lower naive B cells until 12 months after HCT in cGVHD patients.38  These findings are in accordance with results obtained in our study, underscoring the impact of cGVHD on reconstitution of B-cell homeostasis. Of note, Corre et al observed an association of long-term B-cell deficiency with late infections up to 24 months after HCT.38 

During B-cell development, Ig heavy- and light-chain genes are assembled by random gene segment rearrangement resulting in the generation of high diversity but inevitably also in reactivity against self-structures (ie, autoantibody production).39  A first checkpoint for the detection and elimination of such autoreactive B cells represents the pre-B-cell receptor stage, at which surrogate light-chain facilitated B-cell receptor signaling contributes to censoring the development of autoantibody-producing cells.40,41  Immature B cells migrate to the periphery at the transitional B-cell stage, when they are still short-lived and functionally immature. Besides the immature B-cell stage, in the BM the transition between newly emigrant and mature B cells in the periphery is a checkpoint for selection against autoreactive antibodies in humans. Thus, the observed increase of proportions of transitional B cells in cGVHD patients with hypergammaglobulinemia could suggest an increase of circulating autoreactive B-cell proportions because these patients had also a significantly higher frequency of autoantibodies detectable in their serum, which is reminiscent of observations reported in other autoimmune diseases, such as SLE.17  In our study, cGVHD patients with hypergammaglobulinemia had significantly higher BAFF/B-cell ratios resulting from high BAFF levels in the presence of normal CD19+ B-cell numbers compared with patients with normogammaglobulinemia. BAFF and its receptors play a crucial role in peripheral B-cell selection and survival, and increased levels of BAFF have been associated with the development of autoimmune disorders, such as SLE and Sjögren syndrome.42  Besides our findings, Sarantopoulos et al15  and Fujii et al33  reported elevated BAFF levels in cGVHD patients, suggesting inadequate reconstitution of B cells and expansion of activated alloreactive and autoreactive B-cell populations in cGVHD. Recently, Fujii et al reported higher elevations of BAFF, anti-dsDNA antibody, and ANA in patients with late-onset (> 9 months after HCT) compared with early-onset (3-8 months after HCT) cGVHD, suggesting a more predominant B-cell activation in the later stage of the disease.33  Whereas physiologic concentrations of BAFF contribute to purging of autoreactive B cells in the periphery, exogenous administration or overexpression of BAFF is known to rescue autoreactive B cells resulting in production of antichromatin and anti-DNA antibodies.42-45  Indeed, BAFF antagonists are seriously considered for treatment of SLE.46 

Highest BAFF/B-cell ratios were observed in our cGVHD patient cohort with hypogammaglobulinemia, resulting from significantly increased BAFF levels and low CD19+ B-cell numbers. Of note, high serum levels of BAFF, APRIL, and transmembrane activator and calcium-modulating cyclophilin ligand interactor have also been observed in patients with common variable immunodeficiency.47  That lymphopenia might lead to autoimmunity seems to be a more general theme in the immune system and is to some degree reminiscent of the reconstitution phase after T-cell lymphopenia. In this lymphopenic state, especially when regulatory mechanisms responsible for the controlled expansion of the remaining low numbers of T cells are missing, uneven selection leads to an oligoclonal expansion of T cells with a distortion of the peripheral T-cell repertoire and the subsequent possibility of initiating autoimmunity.48  Although overall T-cell numbers were not different between the cGVHD cohorts in our study, cGVHD patients with high proportions of CD19+CD21low immature B cells had significantly lower CD8+CD45RA+ memory T cells (66.7 vs 77.4%, P = .03) and CD4+CD45RA+ naive T cells (7.9 vs 12.6%, P = .03), respectively.

Because currently the pathogenesis of cGVHD is still unclear, different mechanisms impacting on various components of the immune system can be considered after allogeneic HCT, resulting in the different immunologic presentations of cGVHD. Interestingly, patients with lung manifestations of cGVHD had the most pronounced distortion of B-cell homeostasis with highest CD19+CD21low immature B-cell proportions and lowest CD19+CD10CD27CD21high naive B-cell proportions compared with other organ manifestations of cGVHD. Furthermore, CD19+CD27+IgD+ non–class-switched memory B cells were significantly lower in patients with lung manifestations compared with the ones without BO, which is reminiscent of findings described in patients with common variable immunodeficiency and chronic lung disease seen as bronchiectasis, obstruction, and fibrosis.37,49  Currently, little is known regarding the pathogenesis of BO after allogeneic HCT, but alloimmunity seems probable because bronchiolar inflammation is present in lung biopsy specimens and BO occurs in close association with other manifestations of cGVHD.50  Nevertheless, further studies with larger patient numbers are required to obtain more insights into a potential role of B-cell subpopulations in development of cGVHD of the lung. Although not directly addressed in this study, prospective evaluation of 30 patients without cGVHD suggested a usefulness of the B-cell subpopulations tested in this study for distinction between cGVHD and lack of cGVHD in principle. However, a direct comparison of our findings will have to await completion of a currently ongoing large, prospective, longitudinal study.

In conclusion, assessment of different B-cell subpopulations and BAFF/B-cell ratios in cGVHD allowed distinction of different impairments of humoral immunity seen as either immunodeficiency or autoimmunity. These findings are of potential clinical importance for the management of patients with cGVHD and B-cell subpopulations could serve as novel cellular biomarkers for objective diagnosis and monitoring of activity of cGVHD in these patients.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

Preliminary data of this manuscript were presented at the Annual Meeting of the European Group for Blood and Marrow Transplantation, Vienna, Austria, March 2010.

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.

Acknowledgments

The authors thank Dr Peter Bauer, Institute of Medical Statistics of the Medical University of Vienna, for his assistance in statistical analyses and Mrs Karina Schuch for excellent technical assistance.

This work was supported by the European Commission (grant 037703 STEMDIAGNOSTICS) and 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 S.E. performed the clinical research and collected and analyzed data; R.W., U.K., and A.R. performed the flow cytometric analyses; and S.F. performed statistical analyses.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Hildegard T. Greinix, Medizinische Universitaet Wien, Klinik fuer Innere Medizin I, Knochenmarktransplantation, Waehringer Guertel 18-20, A-1090 Vienna, Austria; e-mail: hildegard.greinix@meduniwien.ac.at; and Winfried F. Pickl, Institut fuer Immunologie, Medizinische Universitaet Wien, Borschkegasse 8, A-1090 Vienna, Austria, e-mail: winfried.pickl@meduniwien.ac.at.

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