Citations

Abstract Background: Post-transplant lymphoproliferative disorder (PTLD) occurs in up to 30% of high-risk solid organ transplant recipients and is strongly associated with the intensity and duration of immunosuppression. While Epstein-Barr virus (EBV)-positive monomorphic PTLD and diffuse large B-cell lymphoma (DLBCL) share overlapping morphologic features and expression of canonical B-cell markers, they exhibit distinct EBV transcriptional programs. Immunosuppressive regimens, essential for preventing graft rejection, impair EBV-specific cytotoxic T lymphocyte (CTL) responses, thereby promoting EBV-driven lymphomagenesis. We hypothesized that these differences in viral transcription and immune evasion reflect fundamentally distinct immune microenvironments and host responses in EBV-positive PTLD compared to DLBCL. Methods This prospective, non-interventional study has been conducted at Moffitt Cancer Center since 2019. To characterize tumor-specific T-cell responses in EBV-positive PTLD versus DLBCL, peripheral blood mononuclear cells (PBMCs) from enrolled patients were infected with EBV (ATCC VR-1492) to generate autologous, immortalized lymphoblastoid B-cell lines (LCLs). EBV antigen-specific immune responses were quantified using an IFN-γ ELISpot assay targeting EBNA1, LAMP1, LAMP2A, BARF1, and a CEF peptide pool as a control. To optimize cell viability and functional readout post-thaw, we implemented an overnight resting protocol for PBMCs, which demonstrated superior IFN-γ release compared to the conventional 1-hour rest. Results We present pilot data from 11 consecutive patients (55% male; median age 63.4 years), including 3 with PTLD (2 EBV-positive; 2 monomorphic) and 8 with EBV-negative DLBCL. A total of 12 evaluable PBMC samples were analyzed: 7 pre-treatment, 1 mid-treatment, and 4 post-treatment. Successful generation of autologous LCLs was predominantly achieved using pre-treatment samples, whereas samples collected after B-cell–targeted therapy (Rituximab ± CHOP) showed reduced transformation efficiency. Notably, early pre-treatment samples exhibited a relative enrichment of CD27⁺ memory B cells. Baseline flow cytometric profiling of B cells undergoing LCL transformation revealed low CD19 expression (range <1%–15%). Repeat immunophenotyping at 3 weeks identified residual T cells within cultures, suggesting ongoing B-cell clearance. In matched flow cytometry and ELISpot assays from 7 evaluable patients with pre-treatment samples, EBV-positive PTLD cases demonstrated pronounced overexpression of PD-1 and TIM-3, but not LAG-3, alongside markedly diminished T-cell responses to EBV antigens. This distinct immunophenotypic and functional signature suggests T-cell exhaustion or dysfunction, likely driven by prior immunosuppression, which may impair immune surveillance and facilitate EBV-driven lymphomagenesis. Conclusions Our findings reveal distinct immunologic profiles in EBV-positive monomorphic PTLD compared to EBV-negative DLBCL, characterized by immune exhaustion and impaired T-cell functionality. Specifically, overexpression of PD-1 and TIM-3—but not LAG-3—in EBV-positive PTLD correlated with diminished antigen-specific T-cell responses, as measured by ELISpot, underscoring the role of prior immunosuppression in shaping ineffective tumor-specific immunity. Successful generation of autologous LCLs was predominantly achieved from pre-treatment PBMCs, highlighting the impact of B-cell–depleting therapies (Rituximab ± CHOP) on downstream immune profiling feasibility. The persistence of residual T cells in long-term LCL cultures suggests ongoing B-cell clearance and may reflect previously unrecognized immune dynamics in PTLD. Collectively, these data provide novel insights into the immune landscape of EBV-driven PTLD and support the rationale for therapeutic strategies aimed at reversing T-cell exhaustion—such as immune checkpoint blockade or adoptive T-cell therapies—to restore anti-EBV immunity in transplant recipients.

Abstract Clinical and preclinical research has demonstrated that iPSC-derived NK (iNK) cells have a high therapeutic potential, yet poor understanding of the detailed process of their differentiation in vitro and their counterpart cell development in vivo has hindered therapeutic iNK cell production and engineering. Here we dissect the crucial differentiation of both fetal liver NK cells and iNK cells to enable the rational design of advanced iNK production protocols. We use a comparative analysis of single-cell RNA-seq (scRNA-seq) to pinpoint key factors lacking in the induced setting which we hypothesized would hinder iNK differentiation and/ or functionality. By analyzing key transcription factor regulatory networks, we discovered the importance of TBX21, EOMES, and STAT5A in the differentiation timeline. This analysis provides a blueprint for further engineering new iPSC lines to obtain iNK cells with enhanced functions. We validated this approach by creating a new line of STAT5A-iPSCs which can be differentiated to STAT5A-expressing macrophages with both NK cell and macrophage features such as perforin production, phagocytosis, and anti-tumor functions.