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Background Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis represent a standard first-line treatment for advanced non-small cell lung cancer (NSCLC). However, the development of acquired resistance remains a significant barrier to sustained clinical benefit. V-domain immunoglobulin suppressor of T cell activation (VISTA) has emerged as a key mediator of tumor-induced immunosuppression through modulation of the tumor microenvironment. Accumulating evidence supports the therapeutic potential of VISTA blockade as a strategy to overcome resistance to PD-1/PD-L1-directed immunotherapy.Methods VISTA, FOXP3, and CD4 expression was quantitatively analyzed in 140 NSCLC tumor specimens using Opal-TSA multiplex immunofluorescence (mIF), with survival correlation and ICI response assessment. Flow cytometric analysis of peripheral blood from 40 NSCLC patients (20 anti-PD-1-treated vs 20 untreated controls) quantified VISTA +CD4+ T cells and CD4+CD25+CD127low/- Treg populations. Naïve T cells were stably transfected with pcDNA3.1-VISTA to establish constitutive VISTA overexpression. TGF-β-induced Treg differentiation (FoxP3+) ± VISTA inhibitors were quantified. Anti-PD-1-resistant Lewis lung carcinoma xenografts were generated, and tumor microenvironment dynamics (p-Smad2/3, VISTA, FOXP3) were analyzed by multiplex immunofluorescence and western blotting.Results Opal-TSA multiplex immunofluorescence analysis revealed a significant positive correlation between VISTA and FOXP3 expression in NSCLC tumor tissues (Pearson’s r = 0.564, P < 0.01) ( figure 1). Peripheral blood analysis showed markedly increased frequencies of VISTA+CD4+ T cells (22.24% vs. 5.82%, P < 0.01) and CD4+CD25+CD127low/– regulatory T cells (Tregs; 8.09% vs. 4.18%, P < 0.05) in anti-PD-1-treated NSCLC patients compared to untreated controls. These findings implicate VISTA and Tregs in mediating resistance to immune checkpoint inhibitors (ICIs). In vitro T cell differentiation assays demonstrated that VISTA overexpression significantly enhanced TGF-β-induced Treg (CD4+CD25+FOXP3+) generation by 30% compared to control cells (P < 0.001) (figure 2). This effect was abrogated by VISTA inhibition, restoring Treg differentiation to baseline levels. Furthermore, in anti-PD-1-resistant Lewis lung carcinoma xenografts murine model, tumor tissues exhibited significantly elevated expression of VISTA and TGF-β, increased Smad2/3 phosphorylation, and enhanced intratumoral Treg infiltration relative to control tumors. Collectively, these data indicate that VISTA contributes to anti-PD-1 resistance via the TGF-β/Smad signaling pathway by promoting Treg differentiation.Conclusions The VISTA +/FOXP3+ expression signature predicts poor prognosis in NSCLC patients. VISTA promotes resistance to anti-PD-1 therapy by enhancing TGF-β signaling, thereby facilitating the differentiation and expansion of Treg cells. Notably, pharmacologic inhibition of VISTA reverses this effect, suggesting that VISTA blockade may restore sensitivity to immune checkpoint inhibitors and represents a promising therapeutic strategy to overcome immunotherapy resistance in NSCLC patients.Abstract 944 Figure 1VISTA+FOXP3+ expression and increased Treg infiltration correlate with poor prognosis in NSCLC patients. a-d. Opal-TSA multiplex immunofluorescence staining of VISTA and FOXP3 in NSCLC tumor sections. e-h. Peripheral blood analysis the frequencies of VISTA+CD4+ T cells and CD4+CD25+CD127low/-Tregs in anti-PD-1-treated/control NSCLC patientsAbstract 944 Figure 2VISTA induces PD-1 resistance via TGF-β-dependent Treg immunosuppression. a-b. T cell differentiation assays demonstrated that VISTA overexpression and TGF-β-induced Treg generation c-h. Multiplex immunofluorescence and Western blot analysis of tumor tissues expression of VISTA and TGF-β, p-Smad2/3 across treatment groups