Enhancing of anti-viral activity against HIV-1 by stimulation of CD8+ T cells with thymic peptides

Abstract

HIV-1 can be neutralized by soluble factors produced and secreted by activated CD8+ T cells. Production of such anti-viral CD8 factors (including chemokines) can be induced with IL-2 or phytohaemagglutinin (PHA). In addition to PHA or IL-2, we have co-stimulated CD8+ T cells with PHA/IL-2 and a mixture of thymic peptides (TP) of molecular weights below 10 kD. For the activation, CD8+ T cells were purified from peripheral blood mononuclear cells of HIV-1- individuals and any resultant anti-viral activity was monitored using an HIV-1 neutralization assay. Using HIV-1 isolates highly resistant to chemokine inhibition we detected significantly higher levels of HIV-1 neutralizing activity in CD8+ T cell culture supernatants which had been co-activated with TP. When the TP-induced anti-viral activity was monitored, neutralization of both non-syncytia-inducing (NSI) and syncytia-inducing (SI) patient isolates was enhanced by 38% (NSI, PHA +/- TP), 66% (SI, PHA +/- TP), 28% (NSI, IL-2 +/- TP), and 57% (SI, IL-2 +/- TP) compared with the anti-viral activity present in supernatants from CD8+ T cell cultures stimulated only with PHA or IL-2. Peptide sequence analysis of purified TP showed that the TP mixture predominantly contains peptides with homology to human histone and collagen sequences. Our data demonstrate that CD8+ T cells are additionally activated by a mixture of TP. In this way, the production of HIV-1 neutralizing CD8 factors can be enhanced.

Fig. 1

Features of HIV-1 patient isolates 910 and 952. Both patient isolates were isolated from serum samples of AIDS patients. Virus stocks were grown in donor peripheral blood mononuclear cells (PBMC) and both isolates were characterized by V3 sequencing. The non-syncytia-inducing (NSI) and syncytia-inducing (SI) phenotype and neutralization by RANTES and SDF-1 chemokines were tested in donor PBMC. In bold, positively charged amino acids; underlined, N-glycosylation site.

Fig. 2

CD8⁺ activation and progress of anti-viral activity. Anti-viral activity in supernatants of CD8⁺ T cell cultures was tested after activation of cells with phytohaemagglutinin (PHA; 5 μg/ml, □) or a mixture of PHA and thymic peptides (TP; 0.4 μg/ml, ▪). Anti-viral activity was measured at daily intervals (days 1–5) in a neutralization assay using 25 TCID₅₀ of the non-syncytia-inducing (NSI) patient isolate PI-910. In the neutralization assay virus PI-910 was cultured in PHA- and IL-2-stimulated HIV-1⁻ donor peripheral blood mononuclear cells (PBMC). Each value is the average percent reduction in p24 in triplicate wells; error bars represent s.d.

Fig. 3

Anti-viral activity against syncytia-inducing (SI) and non-syncytia-inducing (NSI) patient isolates. (a) Anti-viral activity in supernatants of CD8⁺ T cell cultures was tested after activation of cells with phytohaemagglutinin (PHA; 5 μg/ml, □) or a mixture of PHA and thymic peptides (TP; 0.4 μg/ml, ▪). (b) Anti-viral activity in supernatants of CD8⁺ T cell cultures was tested after activation of the cells with IL-2 (50 U/ml, □) or a mixture of IL-2 and TP (40 μg/ml, ▪). Each value is the average percent reduction in p24 in triplicate wells; error bars represent s.d.

Fig. 4

Thymic peptide (TP) sequence homologies. All peptides were purified from the crude TP mixture by anion exchange chromatography. *Purified peptides sequenced by combined LC-MS/MS; +, purified peptides sequenced by Edman degradation; −, positions of amino acid identity. Dots represent sequence gaps, numbers the position of the amino acid in the human or bovine sequences from the Genbank database (left column). Sequence homology studies were performed using the Genbank database and the Basic Local Alignment Search Tool (BLAST) from the National Centre for Biotechnology Information (NCBI) [49].