Acute Bone Marrow GvHD Is Associated With Delayed B Cell Neogenesis and Impaired Natural Antibody Response After Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GvHD) and severe infections are main complications limiting the success of allogeneic hematopoietic stem cell transplantion (alloHSCT). Delayed B cell reconstitution followed by B cell immune dysfunction considerably contributes to an increased risk for life-threatening infections. Several studies have shown that B cell regeneration is impaired in patients with systemic GvHD. Bone marrow (BM) suppression is often observed in parallel as GvHD symptoms appear suggesting the BM as a target of GvHD. Thus far, little is known about mechanisms of BM dysfunction during GvHD in alloHSCT patients.

In this study, we investigated the reconstitution kinetics of peripheral blood B cell subsets in adult acute leukemic patients (n=52) before and within six months after alloHSCT by flow cytometry and correlated the data with RT-PCR quantified numbers of kappa-deleting-recombination-excision-circles (KREC), which are stable episomal plasmids generated during BM B cell development. Furthermore, we determined specific B cell antibody responses after in vitrostimulation with CpG, CD40L and T cell cytokines by EliSPOT analysis. To investigate BM as a direct target of allo-reactive T cells we performed histopathological stainings of BM biopsy samples obtained 3-4 weeks after alloHSCT. T cells were detected by specific anti-CD3 antibody staining and osteoblasts were morphologically evaluated.

We observed in all patients a profound B cell immune deficiency already pre-transplant that proceeded within the first months post alloHSCT (mean B cells/ml blood±SEM: 11±3 pretransplant, 3±1 day14, 3±1 day28 post alloHSCT; 83±13 healthy control). Onset of B cell reconstitution is characterized by transitional B cell recovery representing the first B cell subset which emigrates from the BM. B cell reconstitution occurred either early (37% of patients) with a strong increase of transitional B cells between days 60-90 (mean transitional B cells/ml blood±SEM: Day 60, 36±10) or late (33% of patients) with delayed recovering transitional B cells (Day 180, 5±2). KREC copy numbers correlated highly positive and significantly with transitional B cell numbers (Spearman 0.94, p=0.017). Less correlation was obtained with naïve and CD27⁺ memory B cell recovery. Delayed onset of B cell reconstitution was significantly associated with both presence of systemic acute GvHD and full-intensity conditioning therapy (GvHD 71% vs non-GvHD 32%, Fisher´s exact p=0.044; full-intensity 41% vs reduced-intensity 5%, p=0.016). Supporting the hypothesis of bone marrow GvHD we could show a stronger infiltration of CD3⁺ T cells in the BM in late than in early recovering patients (≥5% T cell infiltration: 64% vs 17%, p=0.010). This increased T-cell infiltration was associated with reduced numbers of osteoblasts, known in mice to support B cell lymphopoiesis (no/few osteoblasts: 65% vs 17%, p=0.011). Impaired B cell lymphopoiesis further resulted in a delayed naïve and IgM memory B cell recovery compared to early recovering patients. No recovery of switched-memory B cells was seen for both patient groups within the analyzed time-period. Functionally, ex vivoactivation of patient B cells revealed higher numbers of IgM producing B cells specific for pneumococcal polysaccharide (PnP) at day 180 post alloHSCT in early than in late recovering patients. Polyclonal IgG producing B cells were significantly diminished in all patients.

We conclude from these data, that early onset of B cell reconstitution is characterized by strong increase in regenerating transitional B cells within three months after alloHSCT. Herein, KREC appears as a suitable biomarker to monitor BM B cell output post-transplant. B cell regeneration is significantly delayed in patients showing increased occurrence of systemic acute GvHD and stronger T cell infiltration with loss of osteoblasts in the BM. Thus, delayed onset of B cell reconstitution might result from acute BM GvHD in which alloreactive T cells lead to an osteoblast niche destruction. Increased PnP specific IgM antibody responses are most likely result of higher numbers of early reconstituted transitional and IgM memory B cells but not naïve B cells that were shown not to produce IgM upon CpG stimulation (Capolunghi F et al. 2008). Thus, early B cell reconstitution might provide a first natural antibody immunity after alloHSCT, emphasizing the importance of a functional bone marrow niche.