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`
`TRANSPLANTATION
`
`Altered B-cell homeostasis and excess BAFF in human chronic graft-versus-host
`disease
`Stefanie Sarantopoulos,1 Kristen E. Stevenson,2 Haesook T. Kim,2 Corey S. Cutler,1 Nazmim S. Bhuiya,1
`Michael Schowalter,1 Vincent T. Ho,1 Edwin P. Alyea,1 John Koreth,1 Bruce R. Blazar,3 Robert J. Soiffer,1 Joseph H. Antin,1
`and Jerome Ritz1,4,5
`
`1Division of Hematologic Malignancies and Department of Medical Oncology and 2Department of Biostatistics and Computational Biology, Dana-Farber Cancer
`Institute, Boston, MA; 3Cancer Center and Department of Pediatrics, Division of Bone Marrow Transplantation, University of Minnesota, Minneapolis; 4Cancer
`Vaccine Center, Dana-Farber Cancer Institute, Boston, MA; and 5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA
`
`Chronic graft-versus-host disease (cGVHD)
`causes significant morbidity and mortal-
`ity in patients otherwise cured of malig-
`nancy after hematopoietic stem cell
`transplantation (HSCT). The presence of
`alloantibodies and high plasma B cell–
`activating factor (BAFF) levels in patients
`with cGVHD suggest that B cells play a
`role in disease pathogenesis. We per-
`formed detailed phenotypic and func-
`tional analyses of peripheral B cells in 82
`patients after HSCT. Patients with cGVHD
`Introduction
`
`had significantly higher BAFF/B-cell ra-
`tios compared with patients without
`cGVHD or healthy donors.
`In cGVHD,
`increasing BAFF concentrations corre-
`lated with increased numbers of circulat-
`ing pre–germinal center (GC) B cells and
`post-GC “plasmablast-like” cells, sug-
`gesting in vivo BAFF dependence of these
`2 CD27ⴙ B-cell subsets. Circulating CD27ⴙ
`B cells in cGVHD comprised in vivo acti-
`vated B cells capable of IgG production
`without requiring additional antigen stim-
`
`ulation. Serial studies revealed that pa-
`tients who subsequently developed
`cGVHD had delayed reconstitution of
`naive B cells despite persistent BAFF
`elevation as well as proportional increase
`in CD27ⴙ B cells in the first year after
`HSCT. These studies delineate specific
`abnormalities of B-cell homeostasis in
`patients with cGVHD and suggest that
`BAFF targeting agents may be useful in
`this disease. (Blood. 2009;113:3865-3874)
`
`The limited understanding of the immune mechanisms that result in
`chronic graft-versus-host disease (GVHD) hinders our ability to
`develop effective targeted therapies that would improve the sur-
`vival of patients undergoing allogeneic hematopoietic stem cell
`transplantation (HSCT).1-3 In acute GVHD, tissue injury is medi-
`ated primarily by donor T cells that specifically target minor
`histocompatibility antigens (mHAs) in affected organs.4,5 However,
`despite effective prevention of acute GVHD with agents that
`primarily inhibit T cells (calcineurin inhibitors, mTOR inhibitors,
`and purine analogues),2,6 the incidence and severity of chronic
`GVHD (cGVHD) remain high.1,2 This suggests that
`immune
`mechanisms of cGVHD are distinct from acute GVHD. Studies in
`mHA-mismatched murine transplantation models demonstrate in-
`volvement of B cells in the development of cGVHD.7,8 After
`allogeneic HSCT in humans, alloantibodies to Y chromosome–
`encoded proteins are detectable in 50% of male recipients if they
`received hematopoietic stem cells from a female donor.9 Such
`alloantibodies develop 4 to 9 months after HSCT, and the presence
`of alloantibodies correlated significantly with clinical cGVHD
`development.10 HY antibodies were not associated with acute
`GVHD and did not develop when recipient and donor were sex
`matched. When studied in greater detail, patients with HY antibod-
`ies were also found to have coordinated T-cell responses to distinct
`epitopes in the same HY mHA (DBY).11 These studies have
`suggested that donor B cells play a role in the development of
`cGVHD in humans, and several phase 1 or 2 trials of B cell–
`
`directed therapy with rituximab in steroid-refractory cGVHD have
`revealed clinical responses.12-14 Although these observations pro-
`vide compelling evidence that B cells play an important role in the
`immune pathology of human cGVHD,
`the mechanisms that
`promote and sustain B-cell involvement have not been elucidated.
`HSCT conditioning depletes normal recipient B cells and the
`expansion of normal donor B cells is altered as reconstitution
`occurs in the setting of constant exposure to “foreign” minor
`histocompatibility antigens. Although genetic disparity between
`donor and recipient must exist for cGVHD to develop, sustainable
`autoreactivity occurs after alloreactivity in murine models. Along
`with a more prolonged humoral immune deficiency, a complex
`autoimmune disease–like phenotype is found in cGVHD.15,16 The
`frequent production of autoantibodies in patients with cGVHD17-19
`suggests that disease pathogenesis reflects a critical breakdown in
`peripheral B-cell tolerance after allogeneic HSCT. Furthermore, a
`relative decrease in immature B-cell number in cGVHD compared
`with other post-HSCT patients20 suggests that altered B-cell
`homeostasis is a component of this disease.
`B cell–activating factor (BAFF) plays a critical role in B-cell
`reconstitution and homeostasis after myeloablation.21 Studies of
`B-cell development in murine models have shown that survival of
`normal mature B cells depends on the relative balance of both
`B-cell receptor (BCR) and BAFF signaling.22,23 In addition,
`maintenance of B-cell homeostasis in mice depends on in vivo
`soluble BAFF concentration.24,25 Murine models demonstrate that a
`
`Submitted September 30, 2008; accepted January 11, 2009. Prepublished
`online as Blood First Edition paper, January 23, 2009; DOI 10.1182/blood-
`2008-09-177840.
`
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
`
`The publication costs of this article were defrayed in part by page charge
`
`© 2009 by The American Society of Hematology
`
`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
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`3865
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`3866
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`SARANTOPOULOS et al
`
`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
`
`diverse B-cell pool is required for antigen-induced anergy and
`exclusion of autoreactive B cells from follicular niches.26,27 A set of
`elegant studies subsequently identified a BAFF-mediated B-cell
`tolerance checkpoint in which limiting amounts of BAFF are
`required for ongoing B-cell turnover and avoidance of B-cell
`autoreactivity.28,29 When the pool of normal B cells is reduced,
`excess BAFF promotes survival of autoreactive B cells.29 How-
`ever, even in the presence of high BAFF, high numbers of
`nonautoreactive B cells outcompete autoreactive B cells for avail-
`able soluble BAFF, causing autoreactive B cells to undergo apopto-
`sis.28 Thus, B-cell autoimmunity in transgenic mouse models is
`determined by both the level of soluble BAFF and the numbers of
`competing naive B cells.
`Defective censoring of autoreactive B cells in patients with
`systemic lupus erythematosus (SLE) and rheumatoid arthritis has
`been observed30-32 and high BAFF has been found in patients with a
`variety of autoimmune diseases,33-36 suggesting that excessive
`BAFF stimulation in humans contributes to development of
`autoimmunity. Since high BAFF levels are significantly associated
`with presence of active cGVHD more than 1.5 years after
`HSCT,37,38 we hypothesized that relative B lymphopenia and high
`BAFF after HSCT could support potentially pathologic activated
`alloreactive and autoreactive B-cell populations in cGVHD pa-
`tients. Examining peripheral blood after allogeneic HSCT, we
`found that patients with cGVHD had significantly higher “BAFF/B-
`cell ratio” compared with patients without cGVHD. Serial analysis
`of patients for 1 year after HSCT showed that, in patients without
`cGVHD, BAFF levels gradually normalized as B-cell numbers
`recovered, indicating a normal homeostatic response to B lymphope-
`nia. In contrast, patients who subsequently developed cGVHD
`exhibited delayed reconstitution of naive B cells despite persistent
`BAFF elevation. Using CD27⫹ as a marker of antigen experience
`and ex vivo IgG production as a measure of B-cell activation, we
`found that circulating B cells, including pre-GC B cells, were
`activated in cGVHD. These results suggest that altered B-cell
`homeostasis and excess BAFF contribute to promotion of activated
`B cells in patients with cGVHD.
`
`Methods
`
`Patient characteristics
`
`All patient samples were collected after written informed consent was
`obtained in accordance with the Declaration of Helsinki and approved by
`the Human Subjects Protection Committee of the Dana-Farber/Harvard
`Cancer Center.
`
`Patient group 1
`
`Blood samples for analysis of B-cell reconstitution were obtained from
`57 patients who had undergone allogeneic HSCT between 1994 and 2005
`and were more than 12 months after HSCT. Within this group, BAFF levels
`from 28 samples have been reported previously.37 Clinical characteristics of
`the 57 patients are summarized in Table 1. The study included patients who
`received nonmyeloablative or myeloablative conditioning regimens and
`bone marrow or mobilized peripheral blood stem cell grafts. Patients whose
`primary disease relapsed within 1 year of transplantation and patients
`receiving high-dose steroids (⬎ 0.5 mg/kg prednisone) at
`the time of
`sample collection were excluded. Chronic GVHD status at the time of
`sample collection was categorized according to documented clinical
`examination and laboratory studies using both Seattle criteria and National
`Institutes of Health (NIH) cGVHD consensus criteria. Twenty-two patients
`had “active cGVHD” at the time of primary analysis. This included patients
`with either limited or extensive cGVHD based on clinical severity and
`
`target organs affected per modified Seattle criteria.39 Twenty-three patients
`had “inactive cGVHD” at the time of sample collection. Inactive cGVHD
`was used to describe patients with cGVHD who had achieved a complete
`response to immune-suppressive therapy at the time of analysis. Patients
`who never developed cGVHD after HSCT were designated as having “no
`cGVHD.” Unlike patients with inactive or active cGVHD, patients with no
`cGVHD were receiving no immune-suppressive agents, except one patient
`who received low-dose steroids for a non-GVHD indication (Table 1).
`Median time of analysis was 20.9 months after transplantation for patients
`with active cGVHD compared with 26.6 months after transplantation for
`patients with no cGVHD (P ⫽ .10). Patients with inactive cGVHD were
`significantly later in their post-HSCT course (30.9 months) compared with
`patients with active cGVHD (20.9 months, P ⫽ .007). All other clinical
`characteristics including age, sex,
`type of transplant, and underlying
`hematologic malignancy were similar in patients with active and inactive
`cGVHD. We also studied 33 healthy controls.
`
`Patient group 2
`
`We prospectively followed an additional group of 25 patients from day of
`stem cell transplantation (day 0), with samples obtained every 3 months
`over a 19-month period. These patients received either myeloablative or
`nonmyeloablative conditioning regimens and tacrolimus and/or sirolimus
`as GVHD prophylaxis.6,40 Within this cohort, 8 patients did not develop
`cGVHD by 12 months after HSCT and 17 patients developed cGVHD
`between 3 and 12 months after HSCT. All patients in group 2 were
`analyzed, regardless of prednisone dose or treatment with other immunosup-
`pressive agents. Six of the 17 patients who developed cGVHD had prior
`grades 2 to 4 acute GVHD. None of the patients without cGVHD were
`taking immunosuppressive agents after the 6-month time point. Control
`blood samples were obtained from 8 patients with multiple myeloma after
`the second of a planned tandem autologous transplantation.
`
`Processing of patient plasma and peripheral blood cells
`
`Whole blood was drawn into standard EDTA-containing collection tubes.
`Plasma was separated from whole blood cells by centrifugation at 600g.
`Plasma was stored in aliquots at ⫺80°C and used after first thaw for BAFF
`measurements. Whole blood for flow cytometry studies was also collected
`on day of use in EDTA-containing tubes. Peripheral blood leukapheresis
`products were obtained from 2 cGVHD patients. Discarded leukocyte
`filters from anonymous healthy platelet donors were obtained from the
`Kraft Blood Donor Center at Dana-Farber Cancer Institute (DFCI).
`
`BAFF enzyme-linked immunosorbent assay
`
`Soluble BAFF in patient plasma samples was measured using a
`commercially available enzyme-linked immunosorbent assay (ELISA)
`and the manufacturer’s recommended procedures (R&D Systems,
`Minneapolis, MN).
`
`Flow cytometric analysis of peripheral B cells
`
`Antibodies used for flow cytometry were as follows: CD19 ECD or CD19
`PC7 (both clone J4.119), CD20 ECD (clone B9E9), CD38 PE (clone
`LS198), CD27 PCD5 (clone 1A4), and IgD FITC (clone IA6-2) from BD
`Biosciences Pharmingen (San Diego, CA); BR3-FITC (eBioscience, San
`Diego, CA); and TACI-PE (R&D Systems). Whole blood was processed for
`flow cytometry using the Prep Plus 2 system (Beckman Coulter, Fullerton,
`CA). The lymphocyte gate was established using forward and side scatter.
`A minimum of 50 000 lymphocytes were collected for all samples to ensure
`adequate subset analysis. Gates positive for CD19, IgD, CD38, and CD27
`were first set according to isotype controls. Only CD19⫹ cells were
`analyzed to ensure that CD3⫹ cells were not included in the analysis of
`CD38 or CD27 expression. Anti-CD19 PC7 (Beckman Coulter) was used
`per the manufacturer’s instructions. Red blood cells (RBCs) were lysed and
`leukocytes were fixed using the Beckman Coulter TQPrep system before
`analysis. Cells were analyzed using a Cytomics FC 500 instrument and
`CXP Analysis 2.0 software (Beckman Coulter). To determine BAFF
`receptor profiles, CD19⫹ cells positively selected from large volume
`
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`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
`
`ALTERED B-CELL HOMEOSTASIS IN CHRONIC GVHD
`
`3867
`
`Table 1. Clinical characteristics of 57 patients who underwent allogeneic HSCT
`
`Chronic GVHD
`
`Characteristic
`
`No. (%)
`Median age, y (range)
`Female sex (%)
`Conditioning regimen (%)
`Myeloablative
`Nonmyeloablative
`Cell source (%)
`Peripheral blood stem cells
`Bone marrow
`HLA matching (%)
`Matched, unrelated
`Matched, related
`Mismatched
`Prednisone dose (%)
`0 mg/day
`0-30 mg/day
`Cellcept (%)
`Tacrolimus (%)
`Rapamycin (%)
`Months after transplantation, median (range)
`BAFF level, ng/mL, median (range)
`Grades II-IV aGVHD (%)
`Disease (%)
`AML/AML from MDS
`ALL
`CML
`CLL
`MDS
`NHL
`HL
`MM
`Other
`
`Active
`
`22 (45)
`46 (24-64)
`10 (45)
`
`14 (64)
`8 (36)
`
`19 (86)
`3 (14)
`
`10 (45)
`11 (50)
`1 (5)
`
`9 (30)
`13 (59)
`7 (32)
`8 (36)
`8 (36)
`20.9 (13.6-79.2)
`7.0 (1.6-24.5)
`2 (9)
`
`8 (36)
`2 (9)
`0 (0)
`3 (14)
`4 (18)
`2 (9)
`1 (5)
`0 (0)
`2 (9)
`
`Inactive
`
`23 (36)
`47 (19-66)
`11 (48)
`
`17 (74)
`6 (26)
`
`15 (65)
`8 (35)
`
`13 (57)
`9 (39)
`1 (4)
`
`10 (42)
`13 (57)
`5 (21)
`3 (13)
`3 (13)
`30.9 (16.7-134.6)
`5.5 (1.5-24.3)
`5 (22)
`
`5 (22)
`1 (4)
`9 (39)
`0 (0)
`4 (17)
`1 (4)
`0 (0)
`1 (4)
`2 (9)
`
`No
`
`12 (18)
`44 (25-58)
`4 (33)
`
`10 (83)
`2 (17)
`
`9 (75)
`3 (25)
`
`2 (17)
`10 (83)
`0 (0)
`
`11 (92)
`1 (8)
`0 (0)
`0 (0)
`0 (0)
`26.6 (18.7-93.2)
`3.0 (1.4-4.9)
`2 (17)
`
`2 (17)
`3 (25)
`3 (25)
`0 (0)
`0 (0)
`4 (33)
`0 (0)
`0 (0)
`0 (0)
`
`P
`
`.65
`.73
`
`.52
`
`.26
`
`.11
`
`.01*
`.001
`.52
`
`AML indicates acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CLL, chronic lymphoblastic
`leukemia; NHL, non-Hodgkin leukemia; HL, Hodgkin lymphoma; and MM, multiple myeloma.
`*P ⫽ .101 for active group versus none; P ⫽ .007 for active versus inactive group.
`
`leukapheresis samples (Miltenyi Biotec, Auburn, CA) were stained with
`anti–BAFF-R, BR3 PE (clone 8A7; eBioscience), or biotinylated BMCA or
`TACI (R&D Systems) followed by streptavidin-APC antibody (Invitrogen,
`Carlsbad, CA).
`
`Ex vivo purification of B-cell subsets
`
`For purification of CD19⫹CD27⫹ and CD19⫹CD27⫺ B cells, whole EDTA
`anticoagulated blood (10-12 mL) was obtained from post-HSCT patients or
`healthy individuals. For purification of IgD versus CD38 B-cell subsets (Table 2),
`large volume leukapheresis products from 2 cGVHD patients or leukocyte filters
`from 3 healthy donors were obtained. Lymphocytes were isolated in a sterile
`fashion using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden). CD27⫹ or
`CD27⫺ B cells were sorted by flow cytometry to more than 98% purity (BD
`FACSAria Special Order Cell-Sorting System; Becton Dickinson, San Jose, CA).
`Purified CD27⫹ or CD27⫺ B cells (⬎ 95%) were also obtained using the indirect
`
`magnetic labeling system B Cell Isolation Kit II, human (Miltenyi Biotec). In
`brief, non-B cells were retained on magnetic-activated cell sorting (MACS)
`columns after staining with a cocktail of biotin-conjugated antibodies against
`CD2, CD14, CD16, CD36, and CD43 and binding to streptavidin magnetic
`microbeads. B cells passed through the column and were positively selected with
`CD27 magnetic beads and enriched to more than 95% CD20⫹CD27⫹. IgD
`versus CD38 B-cell subsets were isolated using a BD FACSAria cell sorter (BD
`Biosciences, San Jose, CA).
`
`IgG measurement
`
`B cells purified to more than 98% purity as assessed by CD20 staining were
`incubated in vitro in complete RPMI medium with 100 ng/mL IL-10 (R&D
`Systems); 250 U IL-2 (BD Biosciences); and 0, 25, 250, or 2500 ng/mL
`BAFF (Axxora LLC, San Diego, CA). IgG was measured using the Human
`IgG Elisa Quantitation Kit (Bethyl Laboratories, Montgomery, TX).
`
`Table 2. “Antigen-naive” (naive and transitional) and CD27ⴙ “antigen-experienced” B cells in patients after HSCT
`Active, n ⴝ 22
`Inactive, n ⴝ 23
`No, n ⴝ 12
`B-cell subset
`
`Healthy, n ⴝ 33
`
`Total naive B cells ⴛ1000/L
`P vs none
`Total transitional B cells ⴛ1000/L
`P vs none
`Total CD27ⴙCD19ⴙ B cells ⴛ1000/L
`P vs none
`CD27ⴙ B cells, %
`P vs none
`
`79.8
`.001
`8.1
`.04
`17.5
`.14
`19.0
`.06
`
`99.1
`.001
`8.5
`.04
`25.3
`.37
`8.4
`.21
`
`260.5
`
`28.7
`
`24.9
`
`6.05
`
`89.5
`.001
`14.4
`.02
`68.1
`.01
`27.3
`⬍ .001
`
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`SARANTOPOULOS et al
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`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
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`
`Figure 1. High BAFF levels and low B-cell numbers result in high BAFF/B-cell ratios in patients with cGVHD. Plasma BAFF concentrations and numbers of CD19⫹
`B cells were measured in 3 groups after allogeneic HSCT: 22 patients with active cGVHD, 23 with inactive cGVHD, and 12 who did not develop cGVHD. Results were
`compared with 33 healthy donors. (A) Plasma BAFF concentrations in each patient group after HSCT and healthy donors. (B) Total number of CD19⫹ B cells in each patient
`group and healthy donors. (C) Median BAFF/B-cell ratio for each patient group and healthy donors. The BAFF/B-cell ratio was defined as nanograms of BAFF per 103 CD19⫹
`B cells. Box plots in each figure depict 75th percentile; median and 25th percentile values and whiskers represent maximum and minimum values.
`
`Statistical analyses
`
`For 2-sample comparison of continuous variables, a Wilcoxon rank sum test
`was performed. The Fisher exact test was used to compare categoric
`variables and the Spearman rank test was used for correlation analysis. All
`tests performed were 2-sided and considered significant at the .05 level.
`
`Results
`
`Relative B lymphopenia and high BAFF/B-cell ratio are
`associated with cGVHD
`
`A detailed analysis of plasma BAFF levels and B-cell phenotype
`was carried out in 57 patients who underwent allogeneic HSCT
`more than 12 months ago (Table 1). As shown in Figure 1A, all
`patients after HSCT had significantly higher BAFF levels com-
`pared with healthy individuals. Patients with active cGVHD had
`BAFF levels that were significantly higher than patients without
`cGVHD (no cGVHD; P ⬍ .001) or healthy donors (P ⬍ .001).
`Patients without cGVHD also had higher than normal BAFF levels
`(3.0 ng/mL vs 1.9 ng/mL, P ⫽ .003). Consistent with a previously
`described “surge” in B-cell number after HSCT,20 B-cell numbers
`in patients without cGVHD were significantly higher than normal
`(Figure 1B). In contrast, patients with active or inactive cGVHD
`had significantly lower numbers of total CD19⫹ B cells compared
`with patients without cGVHD (P ⫽ .001 and P ⫽ .01, respec-
`tively). Since BAFF level per B cell has been determined in murine
`models to be a critical determinant of autoreactive B-cell sur-
`vival,28,29 we calculated BAFF/B-cell ratios (level of BAFF per 103
`B cells) for each patient in each post-HSCT group. Figure 1C
`shows that despite higher than normal BAFF levels, supranormal
`B-cell numbers in patients without cGVHD resulted in BAFF/B-
`cell ratios that were similar to those found in healthy individuals. In
`contrast, patients with cGVHD had significantly higher BAFF/B-
`cell ratios compared with other post-HSCT patients and healthy
`individuals (P ⬍ .001 each). As shown in Figure 1A and B, high
`BAFF/B-cell ratios in patients with cGVHD were due to both high
`BAFF levels and B lymphopenia.
`
`Chronic GVHD is associated with decreased numbers of naive
`B cells
`
`In murine models, high BAFF/B-cell ratios promote survival of
`autoreactive B-cell clones in the absence of competition for available
`soluble BAFF by sufficient numbers of antigen-naive B cells.28 We
`
`therefore examined the relative numbers of antigen-naive B cells and
`antigen-experienced B cells in post-HSCT patients (Table 1) and in
`healthy individuals. In this analysis, antigen-naive B cells were de-
`fined as naive (CD19⫹IgDCD38LoCD27⫺) B cells and transitional
`(CD19⫹IgD⫹CD38HiCD27⫺) B cells; antigen-experienced B cells were
`defined as CD19⫹CD27⫹ B cells. As shown in Table 2, patients with
`active cGVHD had low numbers of naive B cells compared with
`patients with no cGVHD (median time after HSCT, 21 months vs
`27 months, respectively, P ⫽ .01). Patients without cGVHD had su-
`pranormal numbers of both naive and transitional B cells (260.5 ⫻ 106/L
`vs 89.5 ⫻ 106/L [P ⫽ .001] and 28.7 ⫻ 106/L in cGVHD vs
`14.4 ⫻ 106/L [P ⫽ .02], respectively). High total numbers of
`circulating recent bone marrow emigrants or transitional cells and
`naive B cells in patients greater than 1 year after HSCT without
`cGVHD suggests increased bone marrow output and prolonged
`survival of naive B cells compared with healthy individuals and
`with patients with cGVHD. In contrast to naive B cells, numbers
`and frequencies of CD27⫹ antigen-experienced B cells were signifi-
`cantly decreased compared with healthy donors but these cells
`were not different among post-HSCT groups (Table 2). In the
`setting of relative naive B lymphopenia, active cGVHD patients
`tended to have higher frequencies of CD27⫹CD19⫹ B cells (19.0%
`in active cGVHD vs 6.1% in patients without cGVHD), but this
`difference did not reach significance (P ⫽ .06).
`
`Identification of circulating pre-GC (IgDⴙCD38HiCD27ⴙ) B cells
`in cGVHD
`
`In humans, CD27 expression identifies antigen-experienced B cells
`committed to plasma cell differentiation.41 CD27⫹ B-cell subsets
`include IgDLo post–germinal center (GC) memory and plasmablast-
`like (PB)/plasma cell–like (PC) populations. CD27⫹ IgD⫹ B-cell
`populations include “IgD⫹ memory” V region–mutated memory
`B cells.42 In addition, the presence of CD27 on IgD⫹ CD38Hi–
`expressing B cells allows distinction of human peripheral transi-
`tional B cells from pre-GC B cells.43 Pre–germinal center cells
`(GCs), also called BM2⬘, GC founder, or preplasmablast cells, are
`IgD⫹CD38HiCD27⫹. These cells are typically found in human
`tonsils and have also been described in peripheral blood in SLE
`patients.44,45 Table 3 summarizes peripheral CD27⫹ B-cell subset
`phenotypes and their corresponding functional characteristics. We
`used multiparameter flow cytometry to determine whether any of
`these distinct circulating CD27⫹ B-cell subsets were more preva-
`lent in cGVHD. As described in “Flow cytometric analysis of
`peripheral B cells” in “Methods,” we first gated on CD19⫹ B cells
`
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`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
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`ALTERED B-CELL HOMEOSTASIS IN CHRONIC GVHD
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`Table 3. Peripheral blood B-cell subsets analyzed in this study
`Cell-surface
`phenotype
`
`Peripheral blood B-cell subset
`
`Table 4. Proportions of CD27ⴙ B-cell subsets in patients after HSCT
`Active,
`Inactive,
`No,
`Healthy,
`n ⴝ 22
`n ⴝ 23
`n ⴝ 12
`n ⴝ 33
`
`CD19ⴙ subset
`
`CD27ⴚ subsets
`Naive B cells
`Transitional B cells42,43
`CD27ⴙ subsets
`IgD⫹ memory B42,45
`Pre-GC (or “GC founder”)42
`Post-GC memory B
`Plasmablast/plasma cell (PB/PC)
`
`IgD⫹CD38LoCD27⫺
`IgD⫹CD38HiCD27⫺
`
`IgD⫹CD38LoCD27⫹
`IgD⫹CD38HiCD27⫹
`IgDLo/⫺CD38LoCD27⫹
`IgDLo/⫺CD38HiCD27⫹
`
`IgDⴙ memory
`P vs none
`Post-GC memory, %
`P vs none
`Pre-GC, %
`P vs none
`PB/PC, %
`P vs none
`
`8.4
`.04
`20.4
`.06
`41.3
`.06
`86.9
`.08
`
`2.8
`.45
`18.4
`.06
`9.7
`.42
`66.4
`.23
`
`2.3
`
`10.8
`
`2.6
`
`36.8
`
`4.1
`.11
`32.8
`⬍ .001
`4.7
`.65
`40.7
`.80
`
`and examined relative levels of surface IgD, CD38, and CD27⫹ on
`these cells. Figure 2 shows representative examples of flow
`cytometric profiles of CD27⫹ B-cell subgroups identified in
`patients after HSCT and in healthy individuals. Table 4 compares
`the median frequency of each CD27⫹ B-cell subset in patients with
`and without cGVHD and in healthy individuals. Consistent with
`previous reports, all post-HSCT patients had fewer post-GC (IgDLo).46
`We found that the post-GC memory (CD27⫹IgDLoCD38Lo) B-cell
`subgroup was less frequent and lower in number in each post-
`HSCT group compared with healthy individuals, whereas all other
`CD27⫹ subsets were not proportionally or numerically different
`
`from normal (Table 4 and data not shown). As shown in Table 4,
`median IgD⫹ naive (CD38Lo) or pre-GC (CD38Hi) percentages in
`all post-HSCT groups were the same, but these populations tended
`to have higher CD27⫹ expression in cGVHD patients compared
`with patients without cGVHD (although differences did not reach
`significance).
`Overall increased CD27⫹ expression on B-cell subsets in the
`active cGVHD group could not be attributed to time after HSCT,
`since the active cGVHD group was closest in time to HSCT
`(median, 20.9 months) compared with the patients with inactive or
`without cGVHD, and therefore would have been expected to have
`
`Figure 2. Flow cytometric gating algorithm demonstrating circulating CD27ⴙ B-cell subsets in patients after HSCT. Gated CD19⫹ B cells were first examined for IgD
`and CD38 expression (far left column). Subsequently, each subset (a-d) was analyzed for CD27⫹ expression according to Sims et al.43 (a*) IgD⫹ Memory B cells are
`IgDHiCD38LoCD27⫹. (b*) Post-GC memory B cells are IgDLoCD38LoCD27⫹. (c*) Pre–germinal center (GC) B cells are IgDHiCD38HiCD27⫹. (d*) Plasmablast or plasma cell–like
`(PB/PC) B cells are IgDLoCD38HiCD27⫹.
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`SARANTOPOULOS et al
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`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
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`Downloaded from https://ashpublications.org/blood/article-pdf/113/16/3865/1307974/zh801609003865.pdf by NEW YORK UNIVERSITY user on 20 January 2020
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`Figure 3. BAFF promotes CD27ⴙ pre-GC and PB/PC subsets in cGVHD. (A) In vivo correlation between soluble BAFF and numbers of CD27⫹ BCR-activated B-cell subsets
`is shown for patients with active cGVHD (top row) and patients with inactive cGVHD (bottom row). The total number of each CD27⫹ B-cell subset (Table 2) and the BAFF level
`(ng/mL) were natural log–transformed. (B) BCMA, TACI, and BAFF-R expression on pre-GC and PB/PC cells from a patient with active cGVHD compared with healthy control.
`Data are representative of 2 independent experiments conducted using 2 cGVHD patients and 2 healthy donors.
`
`the lowest proportion of CD27⫹ B cells (Table 1). Numbers of each
`CD27⫹ B-cell subset were lower than those found in healthy
`individuals with one notable exception. Importantly, active cGVHD
`patients had significantly higher than normal numbers of pre-GC
`cells (2.5 ⫻ 106/L vs 0.89 ⫻ 106/L, respectively, P ⫽ .03) and
`higher frequency of pre-GC B cells in patients with active cGVHD
`compared with healthy individuals (41.3% vs 4.7%, P ⫽ .02).
`Pre-GC cells have not previously been detected in healthy individu-
`als.42,43 In the current study, we detected circulating cells with a
`pre-GC phenotype in 3 (of 33 tested) healthy individuals. However,
`the pre-GC cells in these 3 healthy individuals were unlike cGVHD
`samples tested in that they did not coexpress the follicular marker
`CD10 (data not shown). Thus, we identified a distinct and
`potentially pathologic pre-GC population, similar to circulating
`pre-GC cells identified by other investigators in SLE patients, in
`patients with cGVHD.44,45
`
`Increasing BAFF levels correlate with increasing numbers of
`activated pre-GC and PB/PC cells in cGVHD
`
`To assess whether high BAFF levels were associated with in-
`creased numbers of any circulating B-cell subsets in vivo, we
`examined possible correlations between the BAFF level and total
`B-cell number in each subset for each patient within each
`post-HSCT group. Figure 3A depicts the significant correlations
`found between cell numbers and high BAFF levels in patients with
`active or inactive cGVHD. Increased BAFF levels in patients with
`inactive disease correlated inversely with post-GC memory subset
`numbers (r ⫽ ⫺0.57, P ⫽ .005), possibly reflecting reconstitution
`and turnover of memory B cells despite high BAFF levels in the
`group of patients with clinically inactive cGVHD. Only patients
`
`with active disease had a significant positive correlation between
`BAFF and PB/PC cell number (r ⫽ 0.52, P ⫽ .01). Active and
`inactive cGVHD patients had positive correlations between only
`BAFF and number of CD27⫹ pre-GC cells (r ⫽ 0.5 and r ⫽ 0.49,
`respectively; P ⫽ .02 each), suggesting that BAFF preferentially
`affected these 2 B-cell populations in vivo. Consistent with an
`activated state, BAFF-R expression was relatively low, whereas
`TACI and BCMA expression was increased47 on pre-GC cells in
`cGVHD (Figure 3B). PB/PC cells also expressed higher BCMA
`levels in cGVHD (Figure 3B). Taken together, these results suggest
`that high BAFF in cGVHD patients promotes survival and
`activation of pre-GC and PB/PC cells.
`We then examined qualitative differences in CD27⫹ popula-
`tions in patients with and without cGVHD. Human CD27⫹ B cells
`are antigen experienced and include memory B cells and antibody-
`secreting cells.41 To determine whether CD27⫹ B cells in patients
`with active cGVHD were functionally activated antibody-secreting
`cells in vivo, we measured IgG production of freshly purified
`CD27⫹ B cells from these patients compared with CD27⫹ B cells
`from patients without cGVHD and from healthy donors. As shown
`in Figure 4A, ex vivo–purified CD27⫹ B cells from patients with
`active and inactive cGVHD produce IgG without requiring in vitro
`antigen or BCR stimulation. Constitutive IgG secretion by CD27⫹
`B cells from patients without cGVHD or from healthy donors did
`not occur. Increasing in vitro BAFF concentration also did not
`result in IgG production by CD27⫹ B cells (data not shown). High
`IgG secretion by the PB/PC population occurred without additional
`BAFF (Figure 4B). Although constitutive IgM production by
`pre-GC B cells was found (data not shown), these cells produced
`IgG after addition of BAFF in vitro (Figure 4B). Except for PB/PC
`
`Figure 4. Constitutive production of IgG by CD27ⴙ B cells
`and B-cell subsets in patients with cGVHD. (A) Spontaneous
`IgG production by purified peripheral blood CD27⫹ B cells from
`post-HSCT patients or healthy individuals. (B) IgG production by
`cGVHD patient B-cell subsets after 48 hours with or without
`additional BAFF in vitro. Error bars indicate SD (⫾ mean).
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`BLOOD, 16 APRIL 2009 䡠 VOLUME 113, NUMBER 16
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`ALTERED B-CELL HOMEOSTASIS IN CHRONIC GVHD
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`Downloaded from https://ashpublications.org/blood/article-pdf/113/16/3865/1307974/zh801609003865.pdf by NEW YORK UNIVERSITY user on 20 January 2020
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`Figure 5. Delayed reconstitution of B cells and increased proportion of CD27ⴙ B cells in patients who develop cGVHD. CD19⫹ B-cell number (left y-axis) and BAFF
`levels (right y-axis) were measured every 3 months after HSCT in 3 patient groups. (A) Eight patients who did not develop cGVHD by 12 months. (B) Seventeen patients who
`developed cGVHD during this period. (C) Eight patients who underwent autologous transplantation after myeloablative conditioning. Numbers of patients measured at ea