1313. Evaluation of Antiviral immune responses in Solid organ transplant recipients: Pre-clinical Studies
Session: Poster Abstract Session: Biomarkers and Correlates of Protection
Saturday, October 5, 2013
Room: The Moscone Center: Poster Hall C
  • #1313_IDWposter.pdf (347.9 kB)
  • Background: Infectious complications are an important contributor to morbidity and mortality in solid organ transplant (SOT) recipients. Epstein Barr virus (EBV) infection causes persistent viremia and post transplant lymphoproliferative disease (PTLD) in a proportion of SOT recipients. Currently EBV infection is monitored by nucleic acid amplification tests (viral loads) that have high sensitivity but poor specificity for predicting PTLD. Using markers of immune competence has been suggested as an adjunct to viral loads to improve the specificity. This is relevant, as there are emerging treatment options such as EBV specific cytotoxic T lymphocytes (CTL). Most PTLD in SOT is of recipient origin and so CTL generation has to be based on risk for PTLD. The specific aims of our study are to evaluate Interferon gamma (IFNg) Elispot for diagnostics and generate CTL for therapy.

    Methods: 1) Peripheral blood mononuclear cells (PBMCs) are incubated with immunogenic EBV peptide pools/EBV lysate and IFNg-Elispot is performed. 2) In separate experiments PBMCs are cultured with EBV peptides/lysate in RPMI-1640 with 5% serum and IL15 for 12days. Cells are then rested overnight before stimulation and analyzed for intracellular IFNg and TNF. 3) T cells from PBMCs are also expanded using CD3/CD28 beads (with X-VIVO 15, 5% serum and IL2, IL7) prior to Elispot and CTL generation.

    Results: In consecutive experiments (n=10) we have reproducibly detected ~150 spot forming cells/105PBMCs in healthy volunteers using Elispot (Figure 1), ready to be extended to patient populations.  We have also shown in healthy volunteers (n=3) that EBV CTL could be generated from seropositive individuals in <2 weeks (Figure 2a&2b). In addition we were able to expand T cells (~50 fold) from small volumes of blood samples with out loss of EBV specificity.

    Conclusion: IFNg Elispot could be a useful adjunct to viral loads for monitoring EBV in solid organ transplant recipients. Rapidly generated EBV CTL (< 2 weeks) from seropositive individuals has the potential for clinical translation (Adoptive immunotherapy). Expansion of T cells without loss of EBV specificity addresses the limitation of working with small volume samples in children.

    Shankar Upadhyayula, MD, MRCPCH, Infectious Diseases, Childrens Hospital of Pittsburgh of UPMC, Pittsburgh, PA


    S. Upadhyayula, None

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