Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Abstract

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4 + helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-c enzyme-linked immunospot assay (ELISPOT) assays and HLApeptide tetramer staining. Single-cell cloning resulted in both CD4 + and CD8 + T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8 + and CD4 + T cell epitopes.

CMV-Specific TCR-Transgenic T Cells for Immunotherapy

“…Therefore, we investigated whether CMV-TCR-transduced T cells could be expanded and enriched by Ag-specific stimulation. As Ag-specific stimulators, we used the autologous pp65-expressing mLCL, which presents endogenously processed pp65 epitopes on class I and II HLA molecules (22,32). For comparison, we performed nonspecific stimulation with anti-CD3 Ab, a mix of allogeneic PBMCs derived from three unrelated donors, and the autologous mLCL without pp65 expression.…”

“…Healthy individuals with persistent CMV infection carry large proportions of CMV-specific CD8 ϩ and CD4 ϩ memory T cells (19,20). Many epitopes derived from the immunodominant CMV Ags pp65, IE-1, and others, which bind to various HLA molecules, have been characterized (19,21,22). Several CMV-specific TCR chain sequences, some of which are shared by various donors (public TCRs), are already known (23)(24)(25)(26)(27)(28)(29)(30)(31).…”

“…Previously, we showed that Ag-specific T cell lines and clones can be efficiently generated, expanded, and tested using B cellbased Ag presentation systems (22,32,33). In this study, we used B cells to establish CMV-specific CD8 ϩ T cell clones of various HLA restrictions which recognize endogenously processed CMV Ag.…”

Activated B Cells Mediate Efficient Expansion of Rare Antigen-Specific T Cells

“…For T-cell cloning, YML-specific T cells were enriched from these cultures by staining with PE-labeled YML peptide/HLA-A2 tetramer and anti-PE magnetic beads and separation on paramagnetic columns (Miltenyi, Bergisch Gladbach, Germany). T cell clones were generated by seeding 0.5 T cells per microculture according to standard methods [21].…”

Infection and GVHD

“…However, it may also contribute to GVHD as it has been described for CD4 T cells that induce GVHD via inflammatory signals increasing the expression of HLA class II on non-hematopoietic cells [58]. On the other hand, CD4 T cells are necessary in order to control CMV disease [59] [60,61]. More recently cross-reactive peptides have been described between CMV and minor histocompatibility antigens [54].…”