CⅡTA-mediated up-regulation of MHC reverses immune evasion of leukemia stem cells in refractory/relapsed AML

Abstract

Abstract Background Leukemia stem cells (LSCs) are recognized as the principal reservoir driving chemoresistance and relapse in acute myeloid leukemia (AML). Emerging evidence implicates low expression of major histocompatibility complex (MHC) molecules as a pivotal mechanism by which LSCs evade T-cell surveillance, however, direct functional validation remains limited. Objective To determine how MHC expression levels on LSCs modulate immune escape and to evaluate the therapeutic potential of enforced MHC up-regulation. Methods We used the chemoresistant KG-1a AML cell line CD34+CD38-cells, representing the LSC-enriched population, were isolated from KG-1a using magnetic-activated cell sorting (MACS) to over 99 percent purity, as confirmed by flow cytometry. Lentiviral transduction was performed to generate CⅡTA- de novo expression LSCs (CⅡTA⁺CD34⁺CD38⁻KG-1a) and control cells expressing GFP alone (GFP⁺CD34⁺CD38⁻KG-1a). MHC-I and MHC-II (HLA-A, -B, -C, -DP, -DQ, -DR) expression was assessed by qPCR and Western blot. For in vitro assays, 2×10⁴ target cells were co-cultured with activated CD3⁺T cells at an initial E:T ratio of 1:1 for 7 days, cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, secretion of TNF-α and IFN-γ was quantified by ELISA as indicators of CD8⁺Tcell effector function. In vivo, 16 huPBMC humanized mice were subcutaneously engrafted with 5×10⁶ CⅡTA⁺ or GFP⁺ LSCs (n = 8 mice per group). Upon tumor establishment, mice within each LSC group (CIITA+ or GFP+) were untreated control or doxorubicin treatment (1 mg/kg i.p. every other day) groups. Tumor volume and body weight were monitored daily. After one week, tumors were harvested for H&E histopathology, TUNEL apoptosis staining, and immunofluorescence analysis of CD3, CD4, and CD8 infiltration. Bone-marrow CD3⁺T cells and peripheral CD8⁺T cells were analyzed by flow cytometry for CD4/CD8 ratio, perforin, and CD107a expression. Results CIITA transduction significantly upregulated expression of HLA-B, HLA-C, and HLA-DR (P < 0.01), while expression of HLA-A, -DP, and -DQ was not significantly altered. In vitro, co-culture with CIITA+LSCs resulted in significantly higher levels of LDH release, TNF-α, and IFN-γ compared to co-cultures with either parental KG-1a LSCs or GFP+control LSCs (P < 0.01) In vivo, although tumor volumes and body weights did not differ among groups, the CⅡTA⁺LSC model displayed elevated tumor-cell apoptosis compared with GFP⁺LSC controls (P < 0.05). CD3⁺T cell infiltration was highest in the CⅡTA-overexpressing LSC model, followed by the GFP⁺LSC model, CⅡTA-de novo expression LSC chemotherapy group, and GFP⁺LSC chemotherapy group. In contrast, perforin and CD107a levels in CD8⁺T cells remained comparable across all groups. Conclusion CD34+CD38-KG-1a LSCs utilize low MHC expression to evade immune elimination. CIITA-mediated enhancement of MHC antigen presentation promotes T cell recognition and cytotoxicity, suggesting a promising immunotherapeutic approach to target LSC-driven resistance in relapsed/refractory AML.