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

Abstract

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.

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

“…Two previously described CMV epitope specificities [21,22], restricted through HLA-DRB1*01 and DRB1*13, were confirmed. Our DRB5-restricted T cell clone recognized amino acids 489-507, already described as a presumably DR11-restricted epitope [21,22]. This sequence, which also contains the HLA class I (HLA-A2) epitope NLVPMVATV, can therefore be presented by at least two different class II molecules.…”

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

“…1A). In order to identify a more defined CMV response for our in vitro experiments, we identified three CMV-seropositive healthy volunteers with responses to either CMV-P1 or CMV-P4, which are 15-mer CMV pp65 peptides that have been previously described [24] (Fig. 1A) To ensure that CD4 + CD25 + CD134 + T-cell responses were not being influenced by bystander activation, we stimulated healthy donor whole blood with toxic shock syndrome toxin-1 (TSST-1) superantigen, known to interact almost exclusively with the human T-cell receptor β-chain variable domain 2.1 (TCR Vβ2) [25,26].…”

“…Mononuclear cells were obtained by centrifugation over Ficoll-Paque (GE Healthcare, Buckinghamshire, UK). As part of the RESTORE study, peripheral blood from 50 HIV-infected Thai patients with CD4 + T-cell counts <350 cells/μL was collected before therapy (baseline) and at weeks 4,8,12,24,48 and 96 after ART initiation (ClinicalTrials.gov identifier NCT01296373). At each visit, patients were reviewed and assays were performed on fresh whole blood.…”

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

“…Pentamers were from Proimmune, Oxford, United Kingdom. Protein antigens, derived from different opportunistic pathogens or whole inactivated pathogen bodies, have been described previously (6,7,8,9). Pathogens used as whole bodies or pathogens from which proteins were derived included the following: Candida albicans, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Toxoplasma gondii, Mycobacterium bovis ("Mycobacterium tuberculosis var.…”

“…), cytomegalovirus (CMV), and human immunodeficiency virus type 1 (HIV-1). Peptides were synthesized by INBIOS, Naples, Italy, or by JPT, Berlin, Germany (6,7,8,9). Final antigen concentrations were 10 g/ml for proteins and 1 g/ml for peptides.…”