A Study on the Effect of Luspatercept on Regulatory T Cells and Myeloid Derived Suppressor Cells in Low Risk Myelodysplastic Syndrome with Ring Sideroblasts

In Low-Risk Myelodysplastic Syndromes (LR-MDS), red blood cell transfusion-dependent (RBC-TD) anemia is the primary cause of morbidity and poor outcomes. Luspatecept (Luspa), a recombinant fusion protein that inhibits Smad2/3 signaling induced by TGF-β ligands, is currently approved for the treatment of RBC-TD anemia in LR-MDS with Ring Sideroblasts (LR-MDS-RS). Despite the clinical efficacy of Luspa, its precise mechanism of action is still under investigation. In MDS, immune dysregulation contributes to ineffective hematopoiesis and clonal evolution, with a prominent role played by immune-suppressive populations, primarily represented by Regulatory T-cells (Treg) and Myeloid Derived Suppressive Cells (MDSC). Moreover, Treg-MDSC functional interactions likely involving TGF-B dependent pathways, has been also proposed. By targeting TGF-β signaling pathways, Luspa may indirectly influence the immune microenvironment. Therefore, we aimed to verify if Luspa, by interfering with TGF-β pathway, might affect Treg and MDSCs in patients with LR-MDS-RS.

20 healthy donors and 13 LR-MDS-RS patients with RBC-TD anemia treated with Luspa at Federico II University of Naples have been included in the study from January 2021. Extensive immune profile, including circulating monocyte-MDSC (m-MDSC) and Treg has been performed by multiparametric flow cytometry at baseline and after 6 months of Luspa treatment. Treg were deeply characterized evaluating the expression of the essential transcriptor factor Foxp3 and its highly suppressive isoform containing exon 2 (Foxp3-E2). We performed in vitro experiments to access the ability to Luspa to affect TGF-B dependent Treg induction.

No significantly change in the amount of CD4 and CD8 T cells, B cells, NK cells have been observed when compared to HD. We analyzed CD54 expression, as a marker of antigen-dependent T cell activation, on both CD4 and CD8 T cells. In MDS patients, before treatment, we found an increase of CD54 only in CD4 T cell. After Luspa therapy, we found a significantly increase of CD54 in both CD4 and CD8 T cells compared to HD [p<0.05]. Moreover, after Luspa, CD4 and CD8 show a significantly higher proliferation rate, evaluated through ki67 expression (respectively, M: 3.18, IQR: 1.88-3.91; M: 3.85, IQR: 3.2-8.38), compared to HD (respectively, M: 1.73, IQR: 1.45-2.32; M: 2.23, IQR: 1.6-3.6). Comparing CD54 and ki67 expression before and after Luspa, there is a noticeable upward trend, although it is not statistically significant. Focusing on Treg, the VA of CD4 T cells expressing Foxp3 is significantly decreased in patients at baseline and after Luspa compared to HD (respectively, M: 24.6/µl; IQR: 14.36-35.85/µl; M: 66.67/µl; IQR: 48.62-86.97/µl). However, the VA of highly suppressive Tregs expressing Foxp3-E2 is significantly decreased after Luspa compared to before treatment (respectively, M: 8.76/µl; IQR: 2.34-12.12/µl; M: 11.51/µl; IQR: 9.12-41.37/µl). In patients at baseline, Foxp3-E2/overall Foxp3 percentage ratio is significantly increased compared to HD (respectively, M: 0.88; IQR: 0.37-1.03; M: 0.29; IQR: 0.15-0.9), but it significantly decreases after Luspa (M: 0.32; IQR: 0.14-0.69). Rather, Treg do not show a different proliferation rate after Luspa. To confirm the capability of Luspa to suppress Treg, we performed an in vitro assay showing that Luspa can inhibit the 60% of the TGF-β-induction of Foxp3.

m-MDSC percentage is significantly increased in patients before Luspa compared to HD (respectively, M: 4.3, IQR: 0.86-10.75; M: 0.3, IQR: 0.1-0.41), but it significantly decreases after the treatment (M: 0.52; IQR: 0.47-3.45).

In our cohort, based on IWG 2018 criteria, 8 patients achieved a response. These responders exhibited significantly higher expression of CD54 on CD8 T cells and a lower percentage of m-MDSC before the treatment compared to non-responders (p<0.05).

According to our preliminary data, Luspa treatment might inhibit the amount of immunosuppressive populations of Treg and m-MDSC. Additionally, it enhances the activation and proliferation status of CD4 and CD8 T cell effectors. Therefore, we can presume that Luspa, by interfering with TGF-β pathway, has the potential to modify the dysregulated immunological microenvironment of LR-MDS. The study is currently ongoing with the purpose of validating these findings in a larger cohort and of exploring their implications in MDS clinical management.