A novel SIV gag-specific CD4+T-cell clone suppresses SIVmac239 replication in CD4+T cells revealing the interplay between antiviral effector cells and their infected targets

Abstract

To study CD4+ T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4+ T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8+ T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4+ T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4+ effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4+ T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4+ T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4+ T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model.

T-cell responses to KSHV infection: a systematic approach

“…CD8+ T cells isolated from PMBC typically have a 5-7 months maximum life-span before they senesce and die, and CD4+ T cells persist in culture for a shorter time, typically 4-6 months maximum (MTT unpublished data). To preserve TCR specificities of interest for additional studies we have developed a method to clone TCRs from T cells into a murine retrovirus-based TCR transfer vector to place the TCRs onto other MHC restriction-matched cells [ 16 – 21 ]. To capture the ORF37/p49 response, we cloned the Vα and Vβ regions of TCRs from R 20 Clones 1, 29, and 33 directly into a TCR transfer vector with PCR primers reverse engineered from the CDR3 sequence analysis [ 18 ].…”

Adoptive Transfer of Engineered Rhesus Simian Immunodeficiency Virus-Specific CD8+T Cells Reduces the Number of Transmitted/Founder Viruses Established in Rhesus Macaques

“…CD8 ϩ T cells from Exp-1 were transduced with Gag CM9-6 TCR only, while CD8 ϩ T cells from Exp-2 were transduced with either Gag CM9-6 or Tat SL8-42 TCR. To provide SIV-specific CD4 ϩ T cells to enhance CD8 ϩ antiviral activity in vivo by providing CD4 ϩ T helper function, as suggested by mouse and human studies (39,40), or by direct antiviral activity on infected cells (41), naive CD4 ϩ T cells from Exp-2 were also transduced with the Gag CM9-6 TCR. Due to the lack of an appropriate SIV-specific MHC class II-restricted TCR, we used the Gag CM9-6 MHC class I-restricted TCR to transfer specific antiviral effector function to the CD4 ϩ T cells as demonstrated previously (reference 42 and unpublished results).…”

CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering

“…CXCR5 transduction. Transductions of CD8 T cells with CXCR5 retroviral vector supernatants were carried out as previously described (61). Supernatants containing retroviral vector were loaded onto retronectin (TaKaRa/Clontech, Inc.)-coated six-well non-tissue-coated plates (treated with 20 g/ml) and centrifuged at 2,000 ϫ g for 2 h at 32°C.…”

“…ERK1/2 phosphorylation analysis. CXCR5-mediated ERK1/2 signaling was examined by immunoblotting as previously described (61). Briefly, the levels of ERK1/2 and phosphorylated ERK1/2 (pERK1/2) were measured in lysates produced from 2 ϫ 10 6 CD8 T cells using phosphorylation-specific antibodies following stimulation with 2 g/ml of human CXCL13 (ProSpec) for 3, 15, 30, and 60 min in serum-free medium.…”