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Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution.

CD8 and CD4 lymphocytes control cytomegalovirus (CMV) infection in immunocompetent individuals, while patients with defective cellular immunity are prone to endogenous reactivation of latent CMV or, like seronegative subjects, prone to primary infection. Administration of CMV-specific CD8 lymphocytes was beneficial for immunocompromised hemopoietic stem cell (HSC) graft recipients. Since CD4 cells contribute to expansion of cytotoxic T lymphocytes (CTL), we defined new T(h) peptides on the immunodominant protein pp65 recognized by CD4 cells from HLA-typed subjects, in the perspective of complementing CTL administration with CMV-specific T(h) cells. Screening by ELISPOT on CD4 and CD8 subsets using overlapping peptides identified 10 novel CD4 peptides. To simplify procedures to generate T cell lines, we used a CD4 peptide library for T cell stimulation instead of ill-defined viral lysates, without the requirement of dendritic cells. This library stimulated CMV-specific CD4 cells. In fact, peptide-induced CD4 cells responded to pp65 and to the viral lysate. These cells were also devoid of alloreactivity after one stimulation cycle. Since Good Manufacturing Procedure-grade peptides can be synthesized, culture conditions are simplified and alloreactivity is rapidly lost, these procedures based on peptide stimulation can facilitate implementation of adoptive reconstitution of CD4 responses in immunocompromised patients also in the case when the HSC allodonor is available for generation of the T cell line.