Graft- versus-host disease (GvHD) is the major complication observed after allogeneic hematopoietic stem cell transplant (allo-HSCT). Steroids represent the first-line treatment but approximately one-third of patients are refractory to steroid and have poor outcomes. Novel therapies are needed for this difficult to treat population of patients with steroid-refractory GvHD (SR-GvHD).
This month the GvHD Hub is focusing on novel targets in the treatment of GvHD, here we present the results of a study, recently published in The Journal of Investigative Dermatol ogyby Johanna Strobl and colleagues, evaluating BCL2 expression levels in T cells of patients with GvHD.
Patients who underwent allo-HSCT were sampled in four cohorts:
- Cohort 1, stratified in “no GvHD” (n = 6) and “GvHD developers” (GvHDdev; n = 5) with appearance of GvHD after Day +14 post allo-HSCT. In this cohort, peripheral blood (PB) samples were collected at four time points (-7, 0, +14, and +100) for transcriptome analysis
- Cohort 2, patients with acute GvHD (aGvHD). Samples from PB mononuclear cells (PBMC; n = 6), skin (n = 31), or gastrointestinal (GI) tract (GIT; n = 17) were collected at the time of aGvHD onset
- Cohort 3, patients with chronic GvHD (cGvHD). PBMC (n = 10), skin (n = 21), liver (n = 4) or lung (n = 3) samples were collected at the time of cGVHD onset
- Cohort 4, PB samples were collected from patients with chronic SR-GvHD (SR; n = 9) and patients with healed steroid-sensitive GvHD in the past (non-SR; n = 10)
- Analysis was performed using RNA sequencing, to identify genes dysregulated in GvHD patients, and quantitative PCR to examine the mRNA levels
- Comparison of gene expression in T cells from no GvHD group and GvHDdev group in Cohort 1 revealed a dysregulation of ten genes. Of these, BCL2and PMAIP1were up-regulated on the day of allo-HSCT (Day 0) and remained up-regulated in the GvHDdev group (Day +14 and +100) but not in the no GvHD group
The levels of BCL2 mRNA were compared between Cohort 2 (aGvHD) and Cohort 3 (cGvHD), and healthy controls (non-inflamed respective organ)
- A significant increase in BCL2 mRNA levels was found in PBMC (n = 12; p< 01), skin (n = 23; p< 0.001), and GIT (n = 17) of patients with aGvHD as well as in PBMC (n = 10; p< 0.05) and skin (n = 20; p< 0.001) of patients with cGvHD
- A trend of elevated BCL2 mRNA was also observed in liver (n = 3) and lung (n = 4) of patients with cGvHD
- BCL2 mRNA levels of GIT aGvHD samples (n = 17; p< 05) were higher compared to non-GvHD GI inflammation biopsies (n = 10) after HSCT indicating the potential use of BCL2 expression for diagnosing GIT aGvHD
To evaluate the association between BCL2 mRNA levels at diagnosis and patient outcomes, levels were correlated to patient characteristics, response to steroid treatment and overall outcome of Cohorts 2 (aGvHD) and Cohort 3 (cGvHD)
- Elevated BCL2 mRNA levels in GvHD skin at diagnosis coincided with SR-GvHD (p< 05)
- In the blood of patients with SR-GvHD higher frequencies of BCL2+ leukocytes (p< 01) and BCL2+ CD8+ T cells (p< 0.01) were observed than in patients with non-SR-GvHD
- BCL2 expression was negatively associated to survival post-HSCT (p = 0.042)
- High expression level of BCL2 at the onset of GvHD was predictive of poor prognosis
- An increase in BCL2+ total leukocytes was observed in PB samples from the aGvHD cohort (n = 10) and the cGvHD cohort (n = 16; p< 05) vsno GvHD controls (n = 4). The upregulation of BCL2 was due to an increase in BCL2+ CD4+ in cGvHD patient samples, whereas in the aGvHD samples it was caused by BCL2+ CD8+ T cells and NK cells
- An analysis of skin lesions of aGvHD (n = 18) and cGvHD samples (n = 13) and healthy controls (n = 12) revealed an increase in BCL2-expressing T-, B-, and NK cells in GvHD skin lesions in comparison to controls
- The levels of BCL2 in regulatory T cells (Treg) and conventional T cells were compared between GvHD-affected skin (aGvHD and cGvHD; n = 5 each) and healthy controls (n = 5). Both Treg and conventional T cells expressed higher levels of BCL2 compared to healthy controls
Susceptibility to BCL-2 inhibition
Further analysis revealed an increase in BCL2/BCL2L1 ratio in aGVHD skin samples (n = 11) compared to controls (n = 13), indicating high sensitivity to venetoclax (a BCL2 inhibitor), further supporting the inhibition of BCL2 for the treatment GvHD. To test the effects of venetoclax on cytotoxic T cells in vitro, mixed leukocyte reactions from PBMC of unrelated donors (n = 7) were established and venetoclax was added after five days of culture:
- The effect of venetoclax on T cell apoptosis was dose-dependent
- After 24 hours of treatment, there was a significant decrease in the proportion of CD8+ T cell (of which 79% expressed BCL2) compared to CD4+ T cells (of which only 20% expressed BCL20.
- In vitro , BCL2 inhibition reduces numbers of cytotoxic effector cells by the induction of apoptosis in cells expressing high levels of BCL2
The effect of BCL2 inhibition was also compared between T cells of patients with SR-GvHD and non-SR-GvHD (Cohort 4). After BCL2-inhibition, apoptosis was observed in both CD4+ and CD8+ T cells of patients with SR-GvHD but not in healthy non-SR-GvHD T cells. Also, BCL2-inhibition lead to a favorable increase in the Treg/conventional T ratio.
- The results of this study showed that BCL2 is upregulated in T cells early in GvHD development as well as in cGvHD and SR-GvHD. Over-expression of BCL2 in GvHD-affected organ samples and immune cell subsets was associated with poor outcomes
- There was increased BCL2 expression in GI samples from patients compared to control samples with non-GvHD GI inflammation suggesting the potential use of BCL-2 expression for diagnosing GI GvHD
- The inhibition of BCL2 induced selective apoptosis in allo-reactive CD4+ cells from patients with aGvHD and in both, allo-reactive CD4+ and CD8+ T cells of patients with SR-GvHD. In contrast, there was no induction of apoptosis in T cells from healthy controls
- In summary, BCL2 can be relevant in diagnosis of aGvHD and cGVHD and as a predictive marker of outcome after allo-HSCT
- The authors suggest BCL2 inhibition should be tested as a target in the treatment of patients with SR-GvHD