Overcoming immune escape in EBV-associated PTLD by understanding the two levels of immunosuppression: the tumor microenvironment and iatrogenic immunosuppression

Background

Epstein-Barr virus (EBV) is one of the most common opportunistic pathogens causing morbidity in immunocompromised patients including the development of B-cell lymphomas (e.g. post-transplant lymphoproliferative disease, PTLD). Viral clearance requires the restoration or establishment of an EBV-specific T-cell response, which is impaired in PTLD patients due to the required immunosuppressive treatment (e.g. Tacrolimus, Tac) as well as the tumor microenvironment (TME).

Methods

Serving as in vitro EBV-associated PTLD model, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) were established from >15 healthy EBV-seropositive donors. At different time points of B-LCL generation, their immunomodulatory receptor expression and secretome were analyzed by flow cytometry and multiplex assays. B-LCL-conditioned medium (CM) was collected at selected time points and pooled from 8-12 donors to generate CM_early (B-LCL age 6-8 weeks), CM_int (12-14 weeks) and CM_late (>30 weeks) which were used to mimic the TME of PTLD in T-cell assays. EBV-specific memory T cells were expanded in presence of autologous B-LCLs and the effect of CM or Tac on their phenotype, activation level and functionality was analyzed by flow cytometry.

Results

Longitudinal analysis of immunomodulatory receptor expression by B-LCLs revealed significant upregulation of CD40, CD86 and PD-L1. Cytokine profile analysis revealed a remarkably high inter-individual variability. While the secretion of the pro-inflammatory IL-6 and IL-8 decreased significantly during B-LCL generation, IL-10 secretion was increased in a fraction of intermediate-aged B-LCLs. Due to its known immunosuppressive effect, two different CM_int were generated based on IL-10 concentration (CM_int-hi, CM_int-lo). In all co-cultures of CD3⁺ T cells with irradiated autologous B-LCLs, central memory T cells were the main active subset. Activation and proliferation were markedly inhibited in presence of Tac as well as CM_int and CM_late.

Conclusion

Creating an immunosuppressive TME is a key mechanism of tumor immune escape. Our data indicate longitudinal changes in TME composition during tumor development. Moreover, T-cell activation and proliferation, both basal attributes of T-cell functionality, were reduced in presence of CM as well as Tac. Our results provide insights into how not only Tac but also the TME affects T-cell responses, thus supporting the development of therapeutic strategies to overcome tumor-mediated immune escape.

Offenlegung Interessenkonflikt:

The authors declare no conflict of interest.