Characterization of the Cellular and Molecular Factors Mediating Antigen-Independent Noncytolytic CD8+ T Cell Suppression of HIV-1

CD8+ T cells have a little understood noncytolytic activity that suppresses human immunodeficiency type 1 (HIV-1) replication in an antigen-independent and MHC-unrestricted manner. This activity specifically inhibits transcription of the HIV-1 proviral genome. Little is understood about the molecular nature of the factor(s) mediating this potent antiviral activity of CD8+ T cells. It is known that a factor secreted by CD8+ T cells can suppress the transcription of HIV-1. However, the antiviral mechanism appears most potent with cell-to-cell contact. Previous investigations by several groups into the nature of this secreted factor have been largely based on a presumption that noncytolytic suppression of HIV-1 by CD8+ T cells is exclusively mediated by a soluble protein. Based on several lines of evidence suggesting the specific involvement of cell-contact determinants in eliciting the noncytolytic CD8+ T cell effector function against HIV-1, a novel approach to the problem was utilized based on the hypothesis that a membrane-bound factor is the prime mediator suppressing HIV-1 transcription. In the ensuing investigation, evidence was uncovered demonstrating the existence of a membrane-localized HIV-1 suppressing factor that was secreted as 30-100nm spherical vesicles termed exosomes. Exosomes from a CD8+ T cell line inhibited the replication of R5 and X4 HIV-1 isolates and were shown to specifically suppress of HIV-1 transcription in acute and chronic models of infection. A much greater degree of complexity to the CD8+ T cell secreted antiviral activity was found than a soluble protein alone could account for. The evidence presented in this study suggests that CD8+ T cell suppression of HIV-1 is predominantly mediated by a membrane-bound protein factor that can be cleaved into a soluble isoform with the secreted CD8+ cell antiviral activity being largely exosome-driven. The results presented in this study provide a much more concrete understanding of the mechanisms underlying CD8+ T cells suppression of HIV-1 transcription and outline new approaches to conclusively identifying the molecular factor mediating potent inhibition of the HIV-1 transcritional promoter

Noncytotoxic Suppression of Human Immunodeficiency Virus Type 1 Transcription by Exosomes Secreted from CD8+ T Cells▿

CD8+ T cells display a noncytotoxic activity that suppresses transcription of human immunodeficiency virus type 1 (HIV-1) in an antigen-independent and major histocompatibility complex-unrestricted manner. To date, the precise cellular and molecular factors mediating this CD8+ T-cell effector function remain unsolved. Despite evidence indicating the dependence of the activity on cell-cell contact, the possibility of a membrane-mediated activity that represses transcription from the viral promoter remains unexplored. We therefore investigated whether this inhibition of HIV-1 transcription might be elicited by a membrane-bound determinant. Using a CD8+ T-cell line displaying potent noncytotoxic HIV-1 suppression activity, we have identified a membrane-localized HIV-1-suppressing activity that is concomitantly secreted as 30- to 100-nm endosome-derived tetraspanin-rich vesicles known as exosomes. Purified exosomes from CD8+ T-cell culture supernatant noncytotoxically suppressed CCR5-tropic (R5) and CXCR4-tropic (X4) replication of HIV-1 in vitro through a protein moiety. Similar antiviral activity was also found in exosomes isolated from two HIV-1-infected subjects. The antiviral exosomes specifically inhibited HIV-1 transcription in both acute and chronic models of infection. Our results, for the first time, indicate the existence of an antiviral membrane-bound factor consistent with the hallmarks defining noncytotoxic CD8+ T-cell suppression of HIV-1

Intercellular communication via exosomes

Exosomes are small membrane bound vesicles between 30-100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. They play a role in intercellular communication by transferring proteins, lipids and RNA to recipient cells. The overall aim of this work has been to further investigate the mechanisms by which cells communicate with each other via exosomes. In Paper I we hypothesized that exosomes from human cells could be used as vectors to provide cells with therapeutic RNA. Herein, exogenous short interfering RNAs were successfully introduced into various kinds of human exosomes using electroporation. Flow cytometry, confocal microscopy and northern blot confirmed the presence of siRNA inside the exosomes. The results showed that exosomes from blood plasma could deliver the siRNA to human monocytes and lymphocytes. The siRNA delivered to the target cells was shown to be functional causing selective gene silencing of mitogen activated protein kinase 1. Our results imply that exosomes from human cells could be used as vectors for delivery of therapeutic exogenous nucleic acids to cells. In paper II we investigated if exosomes from activated CD3+ T cells could play a role in an immunological response by conveying signals from their secreting cells to recipient resting T cells in an in vitro autologous setting. The role of these exosomes was explored in IL-2 mediated T cell proliferation. The results showed that neither exosomes nor IL-2 alone could stimulate proliferation in resting T cells. However, exosomes from stimulated T cells together with IL-2 were able to induce proliferation. T cell cultures stimulated with exosomes and IL-2 showed a higher proportion of CD8+ T cells than cultures without exosomes. Moreover, a cytokine array showed significant changes in the levels of cytokines and chemokines when exosomes were present. The results indicate that activated CD3+ cells communicate with resting autologous T cells via exosomes. The main focus in paper III was to study the cellular mechanism by which esRNA is selectively packaged into exosome vesicles during their biosynthesis. Using RNA gel mobility shift assay, we showed the presence of RNA-binding proteins (RBPs) in exosomes. Moreover, we developed a method for the identification of exosomal RBPs able to bind to the esRNA and cellular microRNA. Using this method, we could identify 31 different RBPs in exosomes and 78 in cells. To evaluate the possible role of the identified RBPs in the transfer mechanism of RNA into intraluminal vesicles, five gene transcripts from the identified RBPs were silenced. The results revealed that a selective gene silencing of hnRNPA2B1 caused a reduction of RNA present in the extracellular vesicles. Thus, a novel transport mechanism was suggested for the packaging of esRNA into the exosomes. In conclusion, the studies presented in this thesis have implications for better understanding the RNA and protein transfer mechanism that occurs between cells via exosomes. The described ability of exosomes to deliver exogenous nucleic acids to cells may be of interest in clinical applications e.g. in gene therapy

Noncytolytic CD8+ Cell Mediated Antiviral Response Represents a Strong Element in the Immune Response of Simian Immunodeficiency Virus-Infected Long-Term Non-Progressing Rhesus Macaques.

The ability of long term non progressors to maintain very low levels of HIV/SIV and a healthy state, involves various host genetic and immunological factors. CD8+ non-cytolytic antiviral response (CNAR) most likely plays an important role in this regard. In order to gain a deeper insight into this unique phenomenon, the ability of CD8+ T cells to suppress viral replication in vitro was investigated in 16 uninfected, longitudinally in 23 SIV-infected long-term non-progressing (LTNPs), and 10 SIV-infected rhesus macaques with progressing disease. An acute infection assay utilizing CD4+ cells from MHC-mismatched monkeys to avoid cytolytic responses was employed. The study has identified CNAR as a long-term stable activity that inversely correlated with plasma viral load. The activity was also detected in CD8+ cells of uninfected macaques, which indicates that CNAR is not necessarily a virus specific response but increases after SIV-infection. Physical contact between CD4+ and CD8+ cells was mainly involved in mediating viral inhibition. Loss of this activity appeared to be due to a loss of CNAR-expressing CD8+ cells as well as a reduction of CNAR-responsive CD4+ cells. In contrast, in vitro viral replication did not differ in CD4+ cells from un-infected macaques, CNAR(+) and CNAR(-) LTNPs. A role for transitional memory cells in supporting CNAR in the macaque model of AIDS was questionable. CNAR appears to represent an important part of the immune response displayed by CD8+ T cells which might be underestimated up to now.peerReviewe

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

BACKGROUND: T cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. OBJECTIVE: To determine the mechanism(s) of immune suppression mediated by the nanovesicles. METHODS: T cell tolerance was induced by i.v. injections of hapten conjugated to self antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated for testing in active and adoptive cell transfer models of CS. RESULTS: Tolerance was shown due to exosome-like nanovesicles in the supernatant of CD8(+) suppressor T cells that were not Treg. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains, or possibly whole antibody, allowing targeted delivery of selected inhibitory miRNA-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin deficient JH(-/-) or miRNA-150(-/-) mice that produced non-suppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. CONCLUSIONS: This is the first example of T cell regulation via systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains