Novel Mechanism for Chronic Graft-Versus-Host Disease Suggests New Treatment Paths

By uncovering a central mechanism in chronic graft-versus-host disease (cGVHD), researchers have identified a potential new treatment target for this common and severe complication of allogeneic hematopoietic stem cell transplant (HSCT).

Amy N. Suthers, PhD, presented results on behalf of the Duke research team headed by Stefanie Sarantopoulos, MD, PhD, at the American Society of Hematology's 59th Annual Meeting & Exposition, December 9 to 12, 2017, in Atlanta, GA.

After undergoing allogeneic HSCT to treat hematologic malignancies, approximately 60% of patients develop cGVHD. Among long-term transplant survivors, cGVHD is a major source of morbidity and the leading cause of non-relapse mortality. Treatment options for cGVHD are currently limited to nonspecific therapies that are associated with unpredictable patterns of response.

Under the guidance of Sarantopoulos, Duke researchers have been studying the mechanisms driving cGVHD, with the goal of identifying potential candidates for targeted therapy.

Role of BCR and TLR in autoimmunity
Abnormal B-cell signaling has long been implicated in the pathophysiology of cGVHD. After patients receive an allogeneic HSCT, the cells of their immune system regenerate in the presence of alloantigen. B-cell receptor (BCR) signaling molecules such as Syk and BLNK contribute to a state of pathogenic activation, in which B cells are immediately responsive to these antigens.

The toll-like receptors (TLRs) are a family of endosomal receptors with diverse effects on the pathophysiology of autoimmune disease. Whereas TLR7 promotes the development of autoimmunity, TLR9 protects against autoimmunity by suppressing the activity of TLR7. The TLR7 and TLR9 receptors can also work synergistically with BCR molecules to enhance their respective pro-autoimmunity and anti-autoimmunity effects.

Prior to the current research, the relationship between BCR-TLR signaling and cGVHD had not been well described. The research group focused on 2 hypotheses to clarify these interactions.

First hypothesis: TLR7 responses are increased in cGVHD
Suthers and colleagues hypothesized that TLR7 responses may be increased in patients with cGVHD. To explore this hypothesis, the team examined the behavior of B cells collected from 23 patients at least 12 months following allogeneic HSCT. At the time of B-cell collection, the patients had active cGVHD (n = 8), inactive cGVHD (n = 6), or no cGVHD (n = 9). B cells from healthy donors (n = 5) were also collected for comparison.

All fresh-frozen B cells were cultured for 24 to 48 hours and assessed for measures of B-cell response, including proliferation and gene expression. After exposure to a TLR9 agonist, no significant differences in cell proliferation or TLR9 expression were found between B cells collected from patients with cGVHD and those collected from patients with inactive or no cGVHD. In contrast, B cells collected from patients with cGVHD exhibited a significant increase in TLR7 transcript expression compared with B cells collected from patients with no or inactive cGVHD (P = .047). These findings support the hypothesis that B cells are activated via TLR7 in patients with cGVHD.

Second hypothesis: BCR-TLR synergy contributes to B-cell activation in cGVHD
In the next set of experiments, the research team examined the role of the BCR-TLR axis in driving the pathogenic activation of B cells in the setting of cGVHD.

Unstimulated B cells collected from patients with active cGVHD produced significantly higher levels of interleukin-6 (IL-6), a marker of B-cell response, than B cells collected from patients with inactive or no cGVHD (P = .039). In the presence of a TLR7 agonist, the difference in IL-6 production was even more pronounced; in this setting, B cells collected from patients with active cGVHD produced significantly higher levels of IL-6 than B cells collected from patients with inactive cGVHD (P = .017) or no cGVHD (P = .008).

Additional experiments showed that BCR and TLR7 synergize to increase the proliferation of B cells in patients with active cGVHD. When stimulated to activate both BCR and TLR7, the B cells collected from patients with active cGVHD were significantly more responsive than B cells collected from healthy donors (P = .045) and patients with no cGVHD (P = .015). Therefore, synergistic BCR-TLR7 signaling appears to drive B-cell hyperresponsiveness in patients with active cGVHD. Blocking this pathway may be an effective strategy for preventing or minimizing the severity of cGVHD.

Implications for cGVHD management
Dozens of molecules are known to be involved in synergistic BCR-TLR7 signaling. Of these, the team identified 2 molecules that are potential candidates for targeted therapy: Lyn and IRF5. Expression levels for Lyn, a BCR-signaling molecule, and IRF5, a transcription factor, were significantly higher in B cells collected from patients with active cGVHD than from any other group (Table).

TABLE. BCR-TLR7 Signaling Pathway Molecules: Expression Levels in B Cells by Donor Group

P Value for Expression Level vs Active cGVHD
Active cGVHD
(n = 7)
Inactive cGVHD
(n = 4)
(n = 7)
Healthy Donor
(n = 6)

BCR = B-cell receptor; cGVHD = chronic graft-versus-host disease; IRF5 = interferon regulatory factor 5; TLR7 = toll-like receptor 7.

The next steps for the research team will be gaining a better understanding of how Lyn and IRF5 contribute to B-cell activation and exploring the potential role of these molecules as treatment targets for cGVHD.

Source: Suthers AN, Su H, Anand SM, et al. Increased TLR7 signaling of BCR-activated B cells in chronic graft-versus host disease (cGVHD). Presented at: American Society of Hematology 59th Annual Meeting; December 9-12, 2017; Atlanta, GA. Abstract 75.