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B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways

Jessica L. Allen, Matthew S. Fore, Jenna Wooten, Philip A. Roehrs, Nazmim S. Bhuiya, Todd Hoffert, Andrew Sharf, Allison M. Deal, Paul Armistead, James Coghill, Don A. Gabriel, Robert Irons, Amber Essenmacher, Thomas C. Shea, Kristy Richards, Corey Cutler, Jerome Ritz, Jonathan Serody, Albert S. Baldwin and Stefanie Sarantopoulos

Article Figures & Data

Figures

  • Figure 1

    B cells from patients with cGVHD have increased protein content and cell size. (A) B-cell protein content per million cells in peripheral B cells isolated from patients without active cGVHD (−cGVHD, n = 11) and with active cGVHD (+cGVHD, n = 9). Data are median ± range from 5 independent experiments. *P = .004 (unpaired 2-tailed t test). (B) B-cell size measured by forward scatter (FSC) in peripheral B cells from patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD) after 24 hours of incubation. Left: Histograms of median FSC of a patient without active cGVHD (shaded area, thin line) and with active cGVHD (open area, bold line). Right: Median of FSC quantified in patients without cGVHD (−cGVHD, n = 5) and with cGVHD (+cGVHD, n = 3). Data are median ± range from 2 independent experiments. *P = .004 (unpaired 2-tailed t test).

  • Figure 2

    CD27+ B cells in patients with cGVHD are enlarged compared to CD27 B cells. (A) Cell size analyzed by forward scatter (FSC) of CD27 and CD27+ B-cell subsets from patients with cGVHD incubated in complete media for 24 hours. Left: Histograms show CD27 B cells (shaded area, thin line) and CD27+ (open area, bold line) from a patient with active cGVHD. Right: Median of FSC quantified in CD27 and CD27+ B cells from patients with active cGVHD. Data are median ± range from a single experiment; n = 3. *P = .042 (unpaired 2-tailed t test). (B) Imaging by light microscopy of IgD+ CD38Hi CD27 B cells and IgD+ CD38Hi CD27+ B cells from a patient with cGVHD incubated for 24 hours. Original magnification ×40.

  • Figure 3

    BAFF increases the cellular size of CD27 and CD27+ B cells from patients with cGVHD. (A) CD27 B cells or (B) CD27+ B cells incubated with BAFF for 24 or 48 hours and cell size analyzed by forward scatter (FSC). Left: Histograms represent (A) CD27 or (B) CD27+ B cells from a patient with cGVHD treated with BAFF for 24 hours (shaded area, thin line) and 48 hours (open area, bold line). Right: Median FSC quantified at 24 and 48 hours after BAFF treatment in (A) CD27 and (B) CD27+ B cells from patients with cGVHD. (A) Data are median ± range from a single experiment; n = 3. *P = .009 (unpaired 2-tailed t test). (B) Data are median ± range from a single experiment; n = 3. *P = .011 (unpaired 2-tailed t test).

  • Figure 4

    Patients with cGVHD have decreased B-cell apoptosis. Frequency of apoptotic cells measured by annexin V and propidium iodide (PI) staining in peripheral B cells from patients without cGVHD. (A) Left: Representative dot plot of a patient with cGVHD at 48 hours. The frequency of viable cells defined by the forward (FSC) and side scatter (SSC) characteristics, 51.9%. Right: Frequency of viable cells quantified at 48 hours in patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD). Data are median ± range. −cGVHD, n = 7. +cGVHD, n = 4. *P = .36 (unpaired 2-tailed t test). (B) Left: Representative dot plot of the same patient as in panel A showing the frequency of apoptotic cells defined as annexin V+ PI cells (lower right quadrant, 26.1%). Right: The frequency of apoptotic cells quantified at 48 hours in patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD). Data are median ± range. −cGVHD, n = 7. +cGVHD, n = 4. *P = .032 (unpaired 2-tailed t test).

  • Figure 5

    BAFF promotes survival of CD27 B cells from patients with cGVHD. Frequency of live cells with or without BAFF measured by FSC and SSC in (A) CD27 or (B) CD27+ B cells from patients with cGVHD. (A) Left panel: Dot plots show the frequency of live CD27 B cells from a patient with cGVHD treated with media (44.4%) or BAFF (58.1%) for 48 hours. Right: Percent increase in live CD27 B cells in response to BAFF quantified at 24 and 48 hours. Data are median ± range from a single experiment; n = 3. *P = .009 (unpaired 2-tailed t test). (B) Left panel: Dot blots show the frequency of live CD27+ B cells from a patient with cGVHD treated with media (49.0%) or BAFF (51.1%) for 48 hours. Right: Percent increase in live CD27+ B cells in response to BAFF quantified at 24 and 48 hours. Data are median ± range from a single experiment; n = 3. Not significant, P = .53 (unpaired 2-tailed t test).

  • Figure 6

    Patients with cGVHD have increased B-cell signaling associated with metabolic activation and survival. (A) Phosphorylation of ERK (pERK at Thr 202 and Thr 204) and AKT (pAKT at S473) measured by immunoblot analysis of peripheral B cells from patients with cGVHD (+cGVHD) and without cGVHD (−cGVHD). Expression was controlled by reprobing with the respective nonphosphospecific protein. (B) Normalized B-cell expression of phosphorylated ERK (left) and phosphorylated AKT (right) in patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD). Data are median ± range, pooled from 5 independent experiments. −cGVHD, n = 9 or 10. +cGVHD, n = 8-10. *P = .021 (unpaired 2-tailed t test with Satterthwaite correction). #P = .009 (unpaired 2-tailed t test with Satterthwaite correction). (C) Isoforms of Bim (Bim EL, Bim L, and Bim S) analyzed by immunoblot analysis of peripheral B cells from patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD). Expression was controlled by reprobing for β-actin. (D) Normalized B-cell expression of the long and short isoforms of Bim in patients without cGVHD (−cGVHD) and with cGVHD (+cGVHD). Data are median ± range from 3 independent experiments. −cGVHD, n = 11. +cGVHD, n = 6. *P = .017 (unpaired 2-tailed t test with Satterthwaite correction).

Tables

  • Table 1

    Clinical characteristics of 51 patients after allogeneic HSCT

    Characteristic−cGVHD (N = 35)+cGVHD (N = 16)P
    No. of transplants (%)
        1 transplant31 (89)12 (75).24
        2 transplants4 (11)4 (25)
    Median age, y (range)48 (21-70)49 (31-64).86
    Sex, no. (%) of females13 (37)8 (50).54
    Median time after transplant, mo (range)89 (36-396)64 (32-145).02
    Conditioning regimen (%)
        Myeloblative20 (57)7 (56).55
        Nonmyeloblative15 (43)9 (44)
    Source of graft (%)
        Peripheral blood29 (83)15 (94).41
        Bone marrow6 (17)1 (6)
    HLA matching (%)
        Matched, unrelated14 (40)7 (44).85
        Matched, related16 (46)6 (38)
        Mismatched5 (14)3 (19)
    Immunosuppressive treatment (%)
        None14 (40)2 (13).06
        Prednisone ≤ 50 mg (4-50 mg)/day8 (23)10 (63).01
        MMF2 (6)3 (19).31
        Tacrolimus4 (11)9 (56).001
        Rapamycin1 (3)1 (6).53
        ATG14 (40)9 (56).37
        Alemtuzumab1 (3)0 (0)1.00
    aGVHD grade 1-4 (%)16 (46)9 (56).56
    Disease (%)
        AML/AML from MDS9 (26)9 (56).26
        ALL1 (3)0 (0)
        CML6 (17)0 (0)
        CLL2 (6)1 (6)
        MDS6 (17)2 (13)
        NHL6 (17)2 (13)
        MM1 (3)0 (0)
        AA1 (3)2 (13)
        Other3 (9)0 (0)
    • MMF indicates mycophenolate mofetil; ATG, antithymocyte globulin; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CLL, chronic lymphoblastic leukemia; NHL, non-Hodgkin leukemia; MM, multiple myeloma; and AA, aplastic anemia.