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

Abstract

Recent data reveal an important role for B cells in the pathogenesis of chronic GVHD (cGVHD). Patients with cGVHD have delayed B-cell reconstitution and elevated BAFF to B-cell ratios compared to patients without cGVHD. The mechanisms promoting and sustaining B-cell activation in this disease, however, remain unknown. As BAFF increases murine B-cell metabolism and survival and maintains autoreactive B-cell clones, we performed ex vivo analyses of peripheral B cells from 51 patients who either had or did not have active cGVHD and were greater than 1 year from the time of allogeneic hematopoietic stem cell transplantation. We found that B cells from patients with active cGVHD were in a heightened metabolic state and were resistant to apoptosis. Exogenous BAFF treatment amplified cell size and survival in B cells from these patients. We found significantly increased signaling through ERK and AKT that associated with decreased levels of proapoptotic Bim, suggesting a mechanistic link between elevated BAFF levels and aberrant B-cell survival. Thus, we identify a role for BAFF in the pathogenesis of cGVHD and define B-cell activation and survival pathways suitable for novel therapeutic development in cGVHD.

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⁺ CD38^Hi CD27⁻ B cells and IgD⁺ CD38^Hi 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).