Role of select T helper cell subsets in the development of Epstein-Barr virus-driven lymphoproliferative disease

Background: Post-transplant lymphoproliferative disease (PTLD) is a significant and often fatal complication of organ transplantation, strongly associated with Epstein-Barr virus (EBV). Severe combined immune-deficient (SCID) mice engrafted with human mononuclear cells (PBMCs) from EBV+ donors spontaneously develop human B cell lymphoproliferative disease (LPD) that resembles PTLD. Approximately 20% of EBV+ donors reproducibly develop LPD in 100% of engrafted mice (high-incidence, HI donors), and ~20% of donors never develop EBV-LPD (no-incidence, NI donors) despite all donors being EBV+. This finding suggests that host factor(s) may predispose individuals to develop EBV-LPD. PBMC from HI donors depleted of CD3+/CD4+ T helper (Th) cells do not develop EBV-LPD, suggesting that Th cells function as potential drivers of EBV-LPD. Methods: PBMCs from three HI and three NI donors were engrafted intraperitoneally into SCID mice. At week 4, spleens were harvested and human Th cells were sorted to purity (>95%). Total RNA was isolated from sorted Th cells and gene expression profiles were evaluated by RNA transcriptome analysis. Sorted Th cells from total PBMCs (resting) were used as baseline controls. Human CD45+ cells from mouse spleens were analyzed by mass cytometry (Cytometry Time of Flight, CyTOF) using a multiparametric antibody panel to identify various Th subsets. To identify specific Th subsets essential to the development of EBV-LPD, PBMCs from 3 HI donors were depleted of Th subsets: follicular Th cells (Tfh), regulatory T cells (Treg) or Tfh/Treg cells. Results: RNA Transcriptome data were analyzed using R DESeq2 for differential expression and revealed significant differences in genes associated with Tfh and Treg cell differentiation and function. Th cells sorted from mice engrafted with PBMCs from HI donors expressed high levels of IFNγ receptor, IL6R, MHC class II, FoxP3, PRMT2 and SYK genes. Applying the analysis software viSNE to CyTOF data revealed significant differences in frequency of B cells, Tfh and Treg subsets between HI and NI. Mice engrafted with Treg depleted or Tfh/Treg depleted PBMCs showed significantly improved survival compared to control nondepleted group (p-value: 0.0008, 0.0145, respectively). There was no difference in engraftment efficiency. The Tfh depleted cohort also had improved survival as compared to the control group; however, this result was not statistically significant (p-value: 0.0941). Conclusions: Significant differences in gene expression and immunophenotypic profiles exist between HI and NI donors in this spontaneous model of EBV-LPD. Depletion of Treg and Tfh subsets from PBMCs prior to engraftment significantly enhanced survival. These data suggest that select Th subsets promote EBV-LPD and provide justification for examining strategies to identify patients at risk for developing EBV-LPD in the pretransplant setting. Citation Format: Elshafa H. Ahmed, Claire Hale, Shelby Sloan, Charlene Mao, Xiaoli Zhang, H. Gulcin Ozer, Deepa Subramaniam, Frankie Jeney, Sarah Schlotter, Porsha Smith, Wing Chan, Palak Sekhri, Christoph Weigel, Christopher C. Oakes, Gregory K. Behbehani, Jianhua Yu, Mireia Guerau, Michael A. Caligiuri, Robert A. Baiocchi. Role of select T helper cell subsets in the development of Epstein-Barr virus-driven lymphoproliferative disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4691.