VISTA Is the Predominant 2nd Generation of ICI (Immune Check-Point Inhibitor) in Ph(-) myeloproliferative Neoplasm

Introduction There has been significant progress in immune checkpoint inhibitor (ICI) therapy in using anti-PD-1 and or anti-CTLA-4 in many solid tumor types. However, only a single failed study has been published in treating Ph(-) myeloproliferative neoplasm (MPN) (Mascarenhas et al 2020, Blood 2020, 135, supply, 1-60.). New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA are being investigated and developed in treating other solid tumors. Therefore, to further possible advance ICI therapy in Ph(-) MPN , we measured these 2nd generation ICIs including LAG-3, TIM-3, TIGIT, VISTA in MPN cells.

Material and Methods

Flow cytometric analysis of 2nd ICI Expression 1) on MDSC : blood MNC cells after CD14 ⁺ microbeads (Miltenyi Biotec) isolation , then assayed by flow cytometry using monoclonal antibodies from BD Biosciences, with gating of HLA-DR ^- CD14 ⁺ CD33 ⁺ as M-MDSC (monocytic MDSC) , and HLA-DR ^- CD14 ^- CD33 ⁺ as G-MDSC (granulocytic ). 2) on Different population cells : Expression levels of the 2nd ICIon the CD3 ⁺, CD4 ⁺, CD8 ⁺, CD14 ⁺, CD34 ⁺, CD41a ⁺, and CD71b ⁺ were also assayed by flow cytometry .

Human VSIG3 Fc treatment and T cell activation and proliferation: MNCs were cultured for three days with recombinant human VSIG3- IgG1 Fc chimera (R&D Systems). Recombinant human IgG1 Fc (R&D System)was used as control.The CD3 ⁺ cells were maintained in culture medium plus IL-2 (10ng/ml), and stained with 5uM CFSE according to the manufacturer's protocol (Thermo Fisher). The CD3 ⁺ cells were stimulated with T-Activator CD3/CD28, and combined VSIG3-IgG1 Fc or IgG Fc treated MNCs at 1:2 ratio for further culture of three days. CFSE cell counts were numerated in gated CD4 ⁺ and CD8 ⁺ cells. Data were processed using FlowJo VX.

Results

1) 2nd Generation ICI (2nd ICI) expression on the MDSC a) Percentage of cells . Fig 1,showed no significant difference of the LAG3 , TIGIT , TIM3 on either G-MDSC or M-MDSC , however, VISTA expression was significantly higher in G-MDSC and M-MDSC from MPN patients than in controls. The mean + SE (% of positive cells)for G-MDSC in MPN was ( 23.97 + 6.86 ) and in controls was (0.00 + 0.00) ( P=0.003). For M-MDSC, the MPN was ( 31.56 + 6.62 ) and for controls was ( 1.47 + 1.47) ( p=0.003). b) MFI of the 2nd ICI . Fig 2, showed no significant difference of LAG3 , TIGIT , TIM3 (MFI) on MDSC between MPN patients and controls. However, VISTA ( MFI) was significantly higher in MPN patients than controls, in G-MDSC , with mean + SE in MPN (3085+ 783.6 ) and controls (179.7 + 64.66) ( P<0.0001) as well as in M-MDSC, with mean + SE in MPN ( 4241 +617.7 ) and controls ( 159.7 + 31.29) ( p<0.001).

2) 2nd ICI expression on different cell populations. a) Percentage of cells expressing 2nd ICI. Fig 3, LAG3 , TIGIT , TIM3 on the different population of cells in MPN were not significantly different from controls, but VISTA was significantly higher in MPN patients than controls on the CD3 ⁺, CD14 ⁺, CD34 ⁺cells. CD3 ⁺ cells were with mean + SE in MPN (20.40 + 5.94 ) and controls (0.91 + 0.44) ( P<0.05), CD14 ⁺ cells were with MPN ( 36.86 +0.12 ) and controls ( 0.79 + 0.24) ( p<0.005) and CD34 ⁺ cells were with MPN (24.08 + 10.47) and controls (2.30 +1,28) (P<0.05) , while in CD41 ⁺ and CD71 ⁺ cells VISTA expression was not different in MPN and controls. b) MFI of the 2nd ICI in different population of cells. There was no significant difference in MFI of LAG3 , TIGIT , TIM3, but there was a significant difference in VISTA expression (MFI ) on the CD3 ⁺, CD14 ⁺, CD34 ⁺ cells in MPN patients and controls. MFI of CD3 ⁺ cells were with mean + SE in MPN (3257 + 673.4 ) and controls (457.0 +59.02) ( P<0.05) ; CD14 were in MPN ( 5399 + 994.3 ) and controls ( 879.3 + 325.2 ) ( p<0.005) and CD34 ⁺ Cells were in MPN (6447 + 3785) and controls (327 + 122.4) (P<0.05), while there were no significance between MPN patients and controls of MFI in CD41 ⁺.and CD71 ⁺ cells.controls ( 879.3 + 325.2 ) ( p<0.005) and CD34 ⁺ cells.

3) Human VSIG3 Fc treatment. as shown in Fig 5, VSIG3 , a specific ligand for VISTA , decreased MPN T cells response in MPN patients

Conclusion . We analyzed the 2nd G of ICI in Ph(-) MPN , We found there were a significant VISTA and not the others including LAG3 , TIGIT , TIM3 expression on the MDSC and different cell population. We also demonstrated by adding VISTA specific ligand , T cell response were blunted . Further studies employing the VISTA antibody and siRNA are in progress. This will form the basis of employing anti-VISTA therapy in the future clinical trials of ICI therapy in Ph(-) MPN.