HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/12/1459 04-1633fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by QUARANTA, M. G. Articles by VIORA, M. Search for Related Content PubMed PubMed Citation Articles by QUARANTA, M. G. Articles by VIORA, M.

HIV-1 Nef equips dendritic cells to reduce survival and function of CD8⁺ T cells: a mechanism of immune evasion

Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy

¹ Correspondence: Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299 00161 Rome, Italy. E-mail: viora{at}iss.it

SPECIFIC AIM

The accessory HIV-1 Nef protein is a crucial determinant for viral replication and pathogenesis. During HIV infection, loss of immune control in the setting of a strong and broad HIV-specific T lymphocyte response leads to a lethal outcome through AIDS. Moreover, dysfunction of dendritic cells (DCs) may contribute to the immune suppression associated with AIDS progression. HIV-1 takes advantage of DC biology to facilitate the onset of infection and its dissemination to surrounding permissive cells.

We recently demonstrated that exogenous Nef selectively activates immature DCs manipulating their phenotypical, morphological, and functional developmental program. The present study was designed to evaluate whether Nef, targeting DCs, could be involved in the dysregulation of CD8⁺ T cell responses associated with HIV-1 infection.

PRINCIPAL FINDINGS

1. Nef inhibits alloreactive CD8⁺ T cell priming
We demonstrated that Nef reduced to <50% the expression of HLA class I ABC molecules on the surface of immature DCs. As priming of CD8⁺ T cells requires T cell receptor-mediated recognition of peptide-MHC class I complexes on DCs, we asked whether Nef affects the ability of DCs to stimulate the proliferation of CD8⁺ T cells. When CD8⁺ T cells were stimulated by Nef-treated DCs, their proliferation was significantly reduced (Fig. 1 A). Mature DCs showed a higher CD8⁺ T cell stimulatory capacity.

View larger version (17K): [in this window] [in a new window]   Figure 1. CD8+ T cells primed with Nef-treated DCs exhibit reduced proliferative response, cytolytic activity, and IFN- production. A) An allogeneic MLR with irradiated DCs cultured at different cell numbers with 105 CD8+ T cells was set up. CD8+ T cells were stimulated with allogeneic untreated DCs (), Nef-treated DCs (), or LPS-treated DCs () and proliferative responses were assessed after 5 days by measuring the amount of [3H]thymidine incorporated. Means cpm ± SE from 4 different experiments are shown. Background [3H]thymidine incorporation of CD8+ T cell culture in the absence of DCs remained <1000 cpm. B) CD8+ T cells were primed for 6 days with untreated DCs (), Nef-treated DCs (), or LPS-treated DCs (), and collected and tested for their ability to lyse allogeneic B cells. Means cpm ± SE from 4 different experiments are shown. *P < 0.05 vs. untreated DC+CD8+. C) Analysis of intracellular IFN- production by CD8+ T cells primed as described in panel B. Intracellular IFN- production was analyzed by flow cytometry measuring costaining of the CD8+ and IFN- producing cells. Means ± SE from 4 different experiments are shown. *P < 0.05 vs. untreated DC+CD8+.

We evaluated the functional competence of CD8⁺ T cells primed by Nef-treated DCs. Nef was able to down-regulate the cytotoxic T lymphocyte (CTL) -inducing capacity of DCs (Fig. 1B ). In addition, Nef significantly down-regulated IFN- production as determined by intracellular staining (Fig. 1C ).

2. Nef triggers CD8⁺ T cell apoptosis
As Nef-treated DCs significantly inhibited CD8⁺ T cell proliferation and functional activity, we examined whether Nef-treated DCs affect CD8⁺ T cell apoptosis. We performed a double-staining flow cytometric analysis using FITC-conjugated annexin V and PE-conjugated anti-CD8⁺ mAb. Nef-treated DCs efficiently triggered apoptosis of CD8⁺ T cells; the increase was readily visible after 4 h (Fig. 2 A), reaching peak levels after 24 h of coincubation (Fig. 2A, B ). LPS-treated DCs did not induce CD8⁺ T cell apoptosis at the time points indicated (data not shown). A direct effect of Nef on CD3/CD28-stimulated CD8⁺ T cell apoptosis was not observed (Fig. 2C ). Notably, Nef ensured the survival of DCs after 24 h of treatment (Fig. 2C ).

View larger version (21K): [in this window] [in a new window]   Figure 2. CD8+ T cells primed with Nef-treated DCs become prone to apoptosis. A) CD8+ T cells were cocultured with untreated DCs or Nef-treated DCs. Quantitative evaluation of apoptosis was performed by flow cytometry measuring costaining of CD8+ and annexin V+ cells. Means ± SE from 6 different experiments are shown. B) % of annexin V+/CD8+ T cells cocultured with untreated DCs or Nef-treated DCs for 24 h. Representative dot-plots are shown. C) CD3/CD28-stimulated CD8+ T cells or DCs were treated for 24 h with Nef and the % of annexin V+ cells was measured by flow cytometry. Means ± SE from 3 different experiments are shown.

3. Nef up-regulates TNF- and FasL production by DCs
We evaluated apoptosis induction associated with paraformaldehyde-fixed DCs and DCs culture-conditioned medium. We observed that paraformaldehyde-fixed Nef-treated DCs induced a 1.8-fold increase of apoptosis. Treatment of CD8⁺ T cells with culture-conditioned medium harvested from 24 h Nef-treated DCs increased apoptosis of CD8⁺ T cell (1.5-fold increase). These results suggest that the mediators of Nef-treated DC-associated apoptotic activity are cell membrane-bound and secreted proteins. Therefore, we evaluated which factors are important for the enhancement of CD8⁺ T cell apoptosis by Nef-treated DCs. Nef triggered statistically significant up-regulation of TNF- secretion compared with untreated DCs, and Nef up-regulated TNF- mRNA transcription rapidly, reaching a pick level after 6 h of treatment. We evaluated intracytoplasmic FasL expression by flow cytometry and found that Nef up-regulated endogenous expression of FasL. Nef scored a 4-fold increase of FasL mRNA level compared with untreated DCs after 24 h. Addition of anti-TNF- and/or anti-FasL mAb blocked the Nef-induced apoptosis.

4. Nef-induced CD8⁺ T cell apoptosis is mediated by caspase 8 activation
To test the involvement of TNF-related death ligands in DC-mediated apoptosis of CD8⁺, we measured caspase 8 and caspase 9 activity. We found a marked activation of caspase-8 in CD8⁺ T cells after coculture with Nef-treated DCs, whereas no activation of caspase-9 was found.

CONCLUSIONS

In the present study we outline a novel mechanism by which Nef, hijacking DC functional activity, induces anergy and apoptosis in CD8⁺ T cells. We found that Nef down-regulates DC surface expression of MHC class I and up-regulates molecules known to be critical for apoptosis induction, such as TNF- and FasL. This likely affects CD8⁺ T cell dysfunction and apoptosis via caspase 8 activation (Fig. 3 ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram.

CD8⁺ T lymphocytes play a central role in HIV infection by direct cytolysis of infected cells and by the secretion of factors that suppress viral replication. However, despite the strong CTL response observed for most infected patients, HIV generally fends off immune attack and induces a lethal outcome through AIDS. Apoptosis has been found to play a crucial role in the pathogenesis of AIDS and to increase with the progression of the disease.

DCs play a crucial role in the generation and regulation of immunity and their interaction with HIV is relevant in the pathogenesis of AIDS. Therefore, manipulation of DC biology represents a strategic mechanism for HIV to escape immune attack. Moreover, viral determinants such as Nef and Tat modulate immature DC function to favor virus spread. We previously demonstrated that Nef, triggering Vav-mediated signaling pathway, leads to phenotypical, morphological, and functional differentiation of DCs. These results point up how Nef may enhance CD4⁺ T lymphocyte activation, thus fostering virus dissemination. Here, we enlarged our earlier results monitoring the effect of Nef on DC ability to prime CD8⁺ T cell responses.

The observation that Nef is efficiently internalized by immature DCs led us to evaluate its effects on these cells. DCs pick up extracellular Nef possibly released by virions or by apoptotic HIV-infected lymphocytes in those tissues (i.e., lymph nodes), where high levels of HIV replication are associated with close interaction between DCs and T lymphocytes.

The effects of Nef on immature DCs deserve attention considering that mucosal DCs and blood DC-SIGN⁺ DCs represent the first HIV-1 targets after sexual transmission and transmission via blood, respectively. Hence, we performed an extensive study using immature DCs as the closest model for in vivo primary HIV infection. We found that Nef down-regulates DC surface expression of HLA-ABC molecules involved in the presentation of antigenic peptides to CD8⁺ T cells. This coincides with the reduced ability of DCs to stimulate the proliferation of CD8⁺ T cells. Next, we evaluated the functional competence of CD8⁺ T cells primed by Nef-treated DCs and found that Nef down-regulates the cytotoxic activity and IFN- production by CD8⁺ T cells. Therefore, CD8⁺ T cells primed by Nef-treated DCs may become anergic. This closely fits with CD8⁺ T cell anergy in HIV infection. CD8⁺ T cells may even become prone to activation-induced cell death after antigen presentation by DCs. This may help to explain the accumulation of incompletely mature HIV-specific CTLs in infected individuals. We demonstrated that DCs produce a greater amount of the apoptosis-inducing molecules TNF- and FasL after treatment with Nef and acquire the ability to kill CD8⁺ T cells. We found a marked increase in caspase 8 activation, a downstream indicator of activation of the TNF-family apoptotic pathways.

Overall, our results suggest that Nef subverts DC homeostasis to affect functional competence and survival of CD8⁺ T cells. Nef oversees CD8⁺ T cells’ anergy and apoptosis by modulating the expression and production of key cell signaling receptors and molecules. Thus, Nef assists HIV-1 to control its host, promoting unresponsiveness and cell death of neighboring uninfected cells. In view of an in vivo relevance, our results provide new insight about the role of Nef in HIV-1 pathogenesis: Nef may favor the escape of HIV-1 from immune surveillance by blocking CD8⁺ T cell functional competence. Elucidation of genes induced through Nef signaling in DCs could reveal pathways used by DCs to drive HIV spread and will be critical to identify therapeutic strategies to bias the DC system toward activation of antiviral immunity instead of facilitating virus dissemination. Our findings using in vitro assays may contribute to understand the function, mechanism of action, and cellular partners of Nef to aid the discovery of suitable anti-Nef drugs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1633fje; doi: 10.1096/fj.04-1633fje

This article has been cited by other articles:

				D. Hoffmann, J. Seebach, A. Cosma, F. D. Goebel, K. Strimmer, H. M. Schatzl, and V. Erfle Therapeutic vaccination reduces HIV sequence variability FASEB J, February 1, 2008; 22(2): 437 - 444. [Abstract] [Full Text] [PDF]

				M. G. Quaranta, A. Napolitano, M. Sanchez, L. Giordani, B. Mattioli, and M. Viora HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56dim and CD56bright NK cell subsets FASEB J, August 1, 2007; 21(10): 2323 - 2334. [Abstract] [Full Text] [PDF]

				B. Majumder, N. J. Venkatachari, E. A. Schafer, M. L. Janket, and V. Ayyavoo Dendritic Cells Infected with vpr-Positive Human Immunodeficiency Virus Type 1 Induce CD8+ T-Cell Apoptosis via Upregulation of Tumor Necrosis Factor Alpha J. Virol., July 15, 2007; 81(14): 7388 - 7399. [Abstract] [Full Text] [PDF]

				M. G. Quaranta, B. Mattioli, L. Giordani, and M. Viora The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS FASEB J, November 1, 2006; 20(13): 2198 - 2208. [Abstract] [Full Text] [PDF]

				D. Lewandowski, M. Marquis, F. Aumont, A.-C. Lussier-Morin, M. Raymond, S. Senechal, Z. Hanna, P. Jolicoeur, and L. de Repentigny Altered CD4+ T Cell Phenotype and Function Determine the Susceptibility to Mucosal Candidiasis in Transgenic Mice Expressing HIV-1 J. Immunol., July 1, 2006; 177(1): 479 - 491. [Abstract] [Full Text] [PDF]

				M. Marquis, D. Lewandowski, V. Dugas, F. Aumont, S. Senechal, P. Jolicoeur, Z. Hanna, and L. de Repentigny CD8+ T Cells but Not Polymorphonuclear Leukocytes Are Required To Limit Chronic Oral Carriage of Candida albicans in Transgenic Mice Expressing Human Immunodeficiency Virus Type 1 Infect. Immun., April 1, 2006; 74(4): 2382 - 2391. [Abstract] [Full Text] [PDF]

				A. Iannello, O. Debbeche, E. Martin, L. H. Attalah, S. Samarani, and A. Ahmad Viral strategies for evading antiviral cellular immune responses of the host J. Leukoc. Biol., January 1, 2006; 79(1): 16 - 35. [Abstract] [Full Text] [PDF]

				R. Hegde, Z. Liu, G. Mackay, M. Smith, Y. Chebloune, O. Narayan, and D. K. Singh Antigen Expression Kinetics and Immune Responses of Mice Immunized with Noninfectious Simian-Human Immunodeficiency Virus DNA J. Virol., December 1, 2005; 79(23): 14688 - 14697. [Abstract] [Full Text] [PDF]

				J. Mann, C. N. Patrick, M. S. Cragg, J. Honeychurch, D. A. Mann, and M. Harris Functional Analysis of HIV Type 1 Nef Reveals a Role for PAK2 as a Regulator of Cell Phenotype and Function in the Murine Dendritic Cell Line, DC2.4 J. Immunol., November 15, 2005; 175(10): 6560 - 6569. [Abstract] [Full Text] [PDF]

				B. Majumder, M. L. Janket, E. A. Schafer, K. Schaubert, X.-L. Huang, J. Kan-Mitchell, C. R. Rinaldo Jr., and V. Ayyavoo Human Immunodeficiency Virus Type 1 Vpr Impairs Dendritic Cell Maturation and T-Cell Activation: Implications for Viral Immune Escape J. Virol., July 1, 2005; 79(13): 7990 - 8003. [Abstract] [Full Text] [PDF]

				D. K. Singh, Z. Liu, D. Sheffer, G. A. Mackay, M. Smith, S. Dhillon, R. Hegde, F. Jia, I. Adany, and O. Narayan A Noninfectious Simian/Human Immunodeficiency Virus DNA Vaccine That Protects Macaques against AIDS J. Virol., March 15, 2005; 79(6): 3419 - 3428. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/12/1459 04-1633fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by QUARANTA, M. G. Articles by VIORA, M. Search for Related Content PubMed PubMed Citation Articles by QUARANTA, M. G. Articles by VIORA, M.