Characterization of antigen‐specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65

Abstract

Human cytomegalovirus (HCMV) and adenovirus cause significant morbidity and mortality in immunocompromised hosts undergoing allogeneic stem cell transplantation. We have previously established a procedure for the generation of polyclonal CTL with specificity against adenovirus and HCMV using a recombinant adenovirus encoding the HCMV pp65 protein (RAdpp65). However, specific CTL expanded after in vitro culture steps were subjected to several in vitro restimulations and, depending on the protocol adopted, this could lead to a selection bias, compromising the clinical benefit. To determine which part of the memory repertoire is selected after in vitro restimulation, we have followed the specificity and clonal composition of pp65-peptide-specific CD8 + T cells in HLA-A*201 individuals before and after repeated in vitro restimulation of cells with RAdpp65, combining HLA tetrameric complexes and immunoscope analysis. Tetramer staining showed that, after in vitro restimulation, up to 60% of CD8 + T cells were virus-specific. Immunoscope analysis showed that the predominant TCRBV diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure. Altogether, these results suggest that the use of RAdpp65 to induce CMV-and adenovirus-specific CTL may be appropriate for immunotherapy.

CD28−CD8+ T cells do not contain unique clonotypes and are therefore dispensable

“…BV8 and BV13 are preferentially used by CMV NLV -specific CD8 + T cells as previously shown by us [44] and others [57][58][59][60][61][62]. Monoclonal expansions with the same CDR3 length additionally suggested the occurrence of identical clonotypes in the CD28 + and CD28 − T cells for both BV8 + as well as BV13 + cells.…”

“…Monoclonal expansions with the same CDR3 length additionally suggested the occurrence of identical clonotypes in the CD28 + and CD28 − T cells for both BV8 + as well as BV13 + cells. It has previously been shown that the repertoire of CMV-specific T cells is not altered by in vitro stimulation [58,59].…”

Human CD62L– memory T cells are less responsive to alloantigen stimulation than CD62L+ naive T cells: potential for adoptive immunotherapy and allodepletion

“…Similar to results published by Hamel et al, memory T cells had a distinct TCR repertoire with certain V␤ families dominated by single peaks. 35 This restriction in diversity likely plays a role in the inability of memory T cells to generate a robust proliferative response following aAg exposure. Chen et al 11 were also able to show that V␤ families were different in CD62L ϩ and CD62L Ϫ populations indicating that antigenspecific tolerance was in part caused by clonal deletion or anergy.…”

Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells

“…23 The feasibility of using pp65 expressed in adenovirus, 24,25 retrovirus, 26 or poxvirus 27 to induce robust CTL response has been demonstrated. In a recent phase 1 trial, CMV-seronegative volunteers were vaccinated with an attenuated strain of canarypox virus (ALVAC) expressing CMV pp65, and they developed robust cellular immunity, equivalent to levels measured in CMV-positive individuals.…”