MECHANISM OF NON-CYTOLYTIC INHIBITION OF HIV BY CD8+T CELLS: EVIDENCE SUPPORTING THE ROLE OF SOLUBLE CD8 ANTIVIRAL FACTORS
Inhibition Through HIV Transcription
The inability to recover virus from asymptomatic HIV-infected individuals unless CD8+ T lymphocytes are first removed, led to the discovery of CD8-mediated suppressor responses [2].
When CD8+ effector cells were added back to the assay virus replication was again suppressed in a dose dependant manner. Subsequently, it has been shown [51, 52] that CD8+ T cells from HIV-infected individuals are able to suppress HIV replication in cultured CD4+ cells by a non-cytolytic mechanism that involves a secreted CAF. These studies demonstrated an arrest of HIV replication at the level of viral transcription. This was shown by culturing naturally-infected CD4+ T cells that were actively producing HIV with either autologous CD8+T cells or a 50% dilution of culture fluids derived from these cells, resulting in a greater than 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days of exposure to CD8+ T cells, but required 6 days in the presence of CAF-containing culture fluids to attain similar level of HIV suppression. Northern blot analysis of CD4+ T cell extracts showed that all viral RNA species (unspliced/ single and double spliced) were reduced in quantity accordingly. CAF-containing culture fluids showed a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells. These experiments suggest non-cytolytic inhibition of HIV by CD8+ cell antiviral activity through the suppression of HIV transcription. More recently, it has been shown [53] that the non-cytotoxic anti-HIV response of CD8+ T cells, demonstrable in vitro, does not affect any of the virus replication steps leading to the integration of HIV provirus, but specifically interrupts the expression of viral RNA.HIV Suppression Through STAT1 Activation
Chang TL et al.
[54] showed the CD8 soluble factor-mediated suppression of HIV to occur via STAT1 activation. Their results reveal that CAF-induced expression of interferon regulatory factor 1 (IRF-1), and that the IRF-1 gene induction was STAT1 dependent. Taken together, the results of Chang et al. suggest that CAF activates STAT1, leading to IRF-1 gene induction and inhibition of gene expression regulated by the HIV-1 LTR.Evidence for the Existence of an Antiviral Membrane-Bound Factor for Non-Cytolytic CD8+T Cell Activity
Recently, Chang and colleagues [55] have shown an elegant demonstration of non-cytotoxic suppression of HIV-1 transcription being achieved by exosomes secreted from CD8+ T cells. Despite evidence indicating the dependence of the activity with cell-to-cell contact, the possibility of a membrane-mediated activity repressing transcription from the viral promoter remains unexplored. They, therefore, investigated whether this inhibition of HIV-1 transciption was elicited by a membrane-bound determinant. Using a CD8+ T cell line displaying potent non-cytotoxic HIV-1 suppression activity, they have identified a membrane-localized HIV-1 suppressing activity that is concomitantly secreted as 30-100 nm sized endosome-derived tetraspaninrich vesicles known as exosomes. Purified exosomes from CD8+ T cell culture supernatant non-cytotoxically 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. This is the first powerful demonstration showing the existence of an antiviral membrane-bound factor consistent with the hallmarks defining non-cytotoxic CD8+ T cell suppression of HIV-1.
Other Side of the Coin for CD8 Antiviral Factors in HIV Disease: Novel CD8 Antiviral Mechanisms
Elevated levels of soluble urokinase-type plasminogen activator (uPA) receptor, CD87/u-PAR, can predict survival of HIV+ individuals.
Recently, Wada et al. [56] purified an HIV-1 suppressing activity from the culture supernatant of an immortalized CD8+ T cell clone, derived from an HIV-1 infected long-term nonprogressor, and identified this activity as the amino-terminal fragment (ATF) of urokinase-type plasminogen activator (uPA). ATF is catalytically inactive, but can suppress the release of viral particles from the HIV-1 infected cell lines via binding to its receptor CD87. In contrast, cell proliferation and the secretion of an HIV-1 LTR driven reporter gene product were not affected by ATF. These findings suggest that ATF may inhibit the assembly and budding of HIV-1. Supporting this It has also been confirmed by several studies that a serine protease (uPA) possesses anti-HIV activity, which functions by blocking the release/budding of virions [56, 57]. This anti-HIV activity of uPA is due to an intracellular signaling mediated by its receptor uPAR, but not by its enzymatic activity. At this stage, it is difficult to explain the functional link between findings described in the paper by Mackewicz et al. [58], and recent findings reported previously by Tumne et al. [55], Wada et al. [56], Alfano et al. [57, 59]. But these disparate antiviral activities will be discussed and clarified in detail in the subsequent chapters.Clarifications on the Possible Nature of Soluble Factor-Mediated Antiviral Activity of CD8+ T Cells
Recent studies [60] have shown that CAF activity is different from other antiviral factors (defensins) reported from CD8+ T cells. The alpha-defensin-1 inhibits HIV-1 infection following viral entry but alpha-defensins 1 to 3 are not responsible for the HIV-1 transcriptional inhibition imparted by CAF. A recent report by [61] also demonstrate that alpha-defensins specifically block the initial phase of the HIV infectious cycle and modulate the expression of CD4, a critical receptor in the physiology of T-cell activation.
Further, it has also been shown that the alpha-defensins exhibit anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus.
While they could demonstrate alpha-defensins in neutrophils and monocytes, no evidence was seen for the production of these peptides by CD8+ T cells. Further, no messenger RNA encoding these proteins was detected in purified CD8 T cells, nor did these cells produced intracellular or extracellular alpha-defensin peptides. Furthermore, antibodies specific for human alpha-defensins 1, 2, and 3 did not block the antiviral activity of CAF-active CD8 cell culture fluids. These data suggest that alpha-defensins are not produced by CD8 cells and are not the components of the elusive CAF, but they possess potent anti-HIV activity and are a vital part of soluble anti-HIV arsenal of neutrophils and monocytes [58].CAF Antiviral Activity is Mediated by Protein
In the context of HIV, cell culture studies have shown that three beta chemokines RANTES, MIP-1beta and MIP-1 alpha are known to be important anti-HIV factors produced by CD8+ T cells, which do not kill the infected cells. These beta chemokines can act individually in blocking HIV replication, but the presence of all three in excess of 500 pg to 500 ng is more effective in blocking HIV replication, which works at the level of cell surface inhibition of CCR5 binding to the envelope glycoprotein [62]. Thus, in summary all the cytokines known in the context of HIV disease (IL-1-IL-13, IL15, IL-16, TNF alpha and beta, INF-alpha, beta and gamma, TGF-beta, GM-CSF, G-CSF, RANTES< MIP-1alpha and beta, MCP-1, 2, 3, GRO-alpha and beta, LIF, IP-10 and lymphotactin) lack identity to the elusive CAF factor produced by CD8+ T cells.
Recently, it has been shown that the HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity, suggesting that CAF production may not be restricted to CTL induced during HIV disease [63, 64]. Moreover, CAF may act after reverse transcription, at least for X4 isolate replication inhibition. Geiben-Lynn R [65] performed detailed analysis of CTL as well as bulk CD8+ T lymphocytes from six HIV-1-infected individuals and from six HIV-1-seronegative individuals.
Their kinetic studies showed that secreted suppressive activities of HIV-1-specific CTL and bulk CD8+ T lymphocytes from all HIV-1-infected persons were significantly higher than that of supernatants from seronegative controls. This suppressive activity was blocked by monensin and brefeldin A, was heat labile, and appeared distinct in pattern from that of chemokines (MDC, I-309, MIP-1alpha, MIP-1beta, and RANTES), cytokines (gamma interferon, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor), and interleukins (interleukin-13 and interleukin-16). The suppressive activity was predominant in supernatants in which molecules were size restricted to greater than 50 kDa. Their data provides a functional link between CD8(+) cells and CTL in the non-cytolytic inhibition of HIV-1 and demonstrates that suppression of X4 virus is mediated through protein(s). To date, these are the only mechanisms known for the non-cytolytic inhibition of HIV by CD8+ T cells and the actual identity of protein(s) conferring this activity remains unknown.Does Non-Cytotoxic Activity Mediated by CD8+T Cells Require Interaction with other Cell Types? Possible Role of DCs in the Enhancement of Non-Cytotoxic anti-HIV Responses
Dendritic cells (DCs) have a number of roles in HIV pathogenesis, including initial HIV uptake and transport to lymphoid tissue, stimulating HIV replication in T cells and priming CD4+ and CD8+ T lymphocyte mediated immunity. As a result of HIV-1 infection, DCs are able to infect and subsequently induce anergy or apoptosis of naive T cells, which contributes to the early loss of HIV-specific CD4+ and CD8+ T cells [66].
Recently, Castelli et al. [67] have shown a powerful demonstration suggesting that mature dendritic cells can enhance CD8 cell non-cytotoxic anti-HIV responses, which implies the role of IL-15. It is already known that The CD8 cell non-cytotoxic anti-HIV response (CNAR) is associated with a long-term healthy clinical state in HIV-infected individuals.
With time, the CNAR is reduced in concomitance with HIV disease progression. CD8+T cells from healthy HIV-infected individuals display potent ability to suppress viral replication without killing the infected cells. HIV-infected individuals who progress to HIV disease display diminishing CD8+T cell non-cytotoxic antiviral response (CNAR). The underlying reasons for this profound loss of this vital antiviral activity are not known. One reason for the loss of CNAR could be attributed the decrease in a T helper 1 (TH-1) cell cytokine profile, which is seen to persist over time in HIV+ individuals and this switches to TH-2 type of immune response This response is characterized by the production of interleukin-4 (IL-4), IL-5, and IL-10. TH-1 cell cytokines, in particular IL-2, are vital in sustained maintenance of CNAR, whereas theTH-2 cytokines inhibit its activity. Since IL-2 production by lymphocytes is a consequence of their interaction with dendritic cells (DCs), it is likely that this compromise in the functional aspect of DC could result in the loss of CNAR over time. Supporting this, it already documented that the there is significant decrease in DC numbers
accompanied by functional impairment following HIV infection [67].
Low CD4+ T cell counts can also contribute to profound loss in CD8+ T cell activity and it is known that the ratio between CD4+ and CD8+ T cells is of vital importance in maintaining both CD8 and CNAR activity. Therefore, it appears that the CD40 ligand (CD40L), a surface protein expressed on CD4+ T cells following activation, is present in low levels, which fails to force maturation of DCs via CD40 in order to induce CD8+ cell immune responses, in particular, CNAR. Dendritic cells mature and secrete IL-15 and IL-12 upon activation by CD40L. IL-15 regulates differentiation and expansion, particularly the memory subsets of CD8+ T cells, whereas IL-12 can cause the differentiation of T cells into TH-1 type cells, which are vital for the secretion of large amounts of interferon. This secretion of interferon, in turn, plays a significant role in enhancing CD8+ T cell responses and CNAR. Thus, it is apparent from the data of Castelli et al., that improving DC/T-cell interactions can possibly lead to a better restoration and enhancement of CNAR in HIV- infected individuals.