VISTA contributes to disease progression in high-risk myelodysplastic syndrome

Abstract

Abstract Myelodysplastic syndrome (MDS) is a hematologic disorder characterized by ineffective hematopoiesis and cytopenias, with a risk of progression to secondary acute myeloid leukemia (sAML), particularly in high-risk MDS (HR-MDS). Thus far, the immune mediated mechanisms which determine the phenotypes of HR-MDS are yet to be elucidated. Targeting immune suppression in HR-MDS with immune checkpoint inhibitors (i.e. anti-programmed death therapy) has had limited success, suggesting the presence of other immune suppressive mechanisms in the bone marrow (BM), such as the expression of unique immune checkpoint molecules. To identify these possible immune mechanisms, we first analyzed publicly available datasets of BM from HR-MDS patients. V-domain Ig suppressor of T cell activation (VISTA, also known as PD-1H) is a co-inhibitory checkpoint molecule known to suppress the inflammatory activation of both T cells and myeloid cells. Our group previously reported that myeloid-expressed VISTA is an immune checkpoint which contributes significantly to AML immune evasion. We hypothesized that VISTA expression by monocytes and macrophages in HR-MDS may also be a significant contributor to MDS immune evasion, leading to MDS phenotypes. To assess whether VISTA may contribute to HR-MDS immune evasion, we analyzed publicly available datasets and compared VISTA expression in the BM of HR-MDS patients to healthy controls. We found that VISTA was significantly upregulated in the BM of HR-MDS patients (p = 1.14×10−6). Furthermore, among MDS patients, higher VISTA expression was associated with significantly worse survival (HR 2.1, 95% CI 1.1-4.0, p = 0.022). Next, we assessed VISTA expression on immune cell subsets in cryopreserved MDS BM aspirate specimens using multi-color spectral flow cytometry. Interestingly, we demonstrated that higher monocyte/macrophage VISTA expression was significantly associated with decreased frequencies of CD4+ T cells (r = −0.77, p = 0.02). Meanwhile, monocyte/macrophage VISTA expression was positively correlated with regulatory T cells (r = 0.74, p = 0.04), suggesting that monocyte/macrophage-expressed VISTA may contribute to an immune suppressive BM microenvironment in HR-MDS. To determine whether monocyte/macrophage-expressed VISTA facilitates immune suppression and disease progression in HR-MDS, we utilized the NUP98-HOXD13 transgenic (NHD13Tg) murine MDS model. NHD13Tg mice develop disease resembling human HR-MDS, with a majority progressing to acute leukemia at around 8-12 months of age. We assessed serial complete blood counts (CBC) in a cohort containing NHD13Tg and VISTA-deficient (VISTA−/−) mice. NHD13TgVISTA−/− mice displayed a delayed onset of anemia compared to NHD13Tg mice (hemoglobin at 5 months: 14.0 g/dL vs. 10.8 g/dL, p = 0.04), indicating slower disease progression. We assessed the BM and spleen of these mice at 7 months. NHD13TgVISTA−/− mice had an increase in monocyte/macrophage frequency compared to NHD13Tg mice (2.9-fold change [4.4% vs. 1.5%] within CD45+ BM cells, p = 0.03; 1.6-fold change [5.3% vs. 3.3%] within CD45+ splenocytes, p = 0.06). In addition, NHD13TgVISTA−/− mice trended towards an increase in CD4+ T cells compared to NHD13Tg mice (6.2-fold change [0.099% vs. 0.016%] within CD45+ BM cells, p = 0.13; 2.9-fold change [3.2% vs. 1.1%] within CD45+ splenocytes, p ≤ 0.05). These findings suggest that genetic deletion of VISTA in NHD13Tg mice may contribute to increased immune activation and reflects human HR-MDS BM. To investigate whether VISTA contributes to the transformation of MDS into sAML, we followed a separate cohort of NHD13Tg and NHD13TgVISTA−/− mice for 12 months. Beginning around 7 months of age, some mice began to display elevated white blood cell (WBC) counts on CBC (WBC: 32.6 K/uL vs. median of 4.8 K/uL). Flow cytometric analysis of peripheral blood, BM, and spleen revealed c-Kit+ blasts, suggesting an onset of acute leukemia. Interestingly, NHD13TgVISTA−/− mice displayed delayed progression to leukemia compared to NHD13TgVISTA+/+ mice, suggesting VISTA as a possible contributing factor to the leukemic transformation of HR-MDS. Taken together, our studies suggest that VISTA may contribute to disease progression and leukemic transformation in HR-MDS by promoting immune evasion. Continued investigation will allow us to better elucidate the role of VISTA in MDS and may establish VISTA as a possible target of future therapies for high-risk MDS.