Characterization of HIV trafficking and DC-SIGN-mediated cytoskeleton rearrangement en the context of HIV transfer across dentritic cells-T lymphocytes infectious synapses

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Thesis

Reference

Characterization of HIV trafficking and DC-SIGN-mediated cytoskeleton rearrangement en the context of HIV transfer across

dentritic cells-T lymphocytes infectious synapses

NIKOLIC, Damjan

Abstract

La transmission de l'infection par le VIH s'effectue principalement par voie sexuelle et la compréhension des mécanismes liés à ce transfert au niveau des muqueuses est un sujet de recherche primordial afin de développer des outils thérapeutiques à même de prévenir la propagation du VIH. Un rôle majeur des cellules dendritiques muqueuses dans la transmission du VIH aux lymphocytes T a déjà été proposé précédemment. Ce travail de thèse a permis de caractériser les mécanismes de transfert de l'infection VIH des cellules dendritiques aux lymphocytes T via les synapses infectieuses. Les éléments présentés dans cette thèse apportent des arguments essentiels dans la compréhension des évènements impliqués au niveau de la synapse infectieuse. Nous démontrons ici le rôle de l'activation de la GTPase Cdc42 et des filopodes dans la transmission du VIH aux lymphocytes T. Nous proposons également que l'activation de la GTPase Cdc42 survient suite à la liaison de l'enveloppe du VIH à DC-SIGN et via les kinases SRC. Nos résultats objectivent également que différentes méthodes prévenant la formation [...]

NIKOLIC, Damjan. Characterization of HIV trafficking and DC-SIGN-mediated

cytoskeleton rearrangement en the context of HIV transfer across dentritic cells-T lymphocytes infectious synapses. Thèse de doctorat : Univ. Genève, 2012, no. Sc. Méd. 7

URN : urn:nbn:ch:unige-214659

DOI : 10.13097/archive-ouverte/unige:21465

Available at:

http://archive-ouverte.unige.ch/unige:21465

Disclaimer: layout of this document may differ from the published version.

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Section de Médecine Fondamentale Département de Microbiologie et Médecine Moléculaire

Thèse préparée sous la direction du Professeur Vincent PIGUET

CHARACTERIZATION OF HIV TRAFFICKING AND DC-SIGN- MEDIATED CYTOSKELETON REARRANGEMENT IN THE CONTEXT OF HIV TRANSFER ACROSS DENDRITIC CELLS-T

LYMPHOCYTES INFECTIOUS SYNAPSES

Thèse

présentée à la Faculté de Médecine de l'Université de Genève

pour obtenir le grade de Docteur en Sciences Médicales « MD-PhD » par

Damjan NIKOLIC

de

Bassecourt (Jura)

Thèse n° 7

Genève 2012

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Formulaire à joindre aux exemplaires de la thèse à remettre pour le dépôt légal

THESE

Informations indispensables à dactylographier

Nom et Prénom : NIKOLIC Damjan

Adresse : Route du Moulin-Roget 51 / 1237 Avully

Faculté : Médecine Département (selon structures officielles) : Microbiologie et Médecine Moléculaire

Directeur de thèse : Pr Vincent PIGUET

Références bibliographiques :

CHARACTERIZATION OF HIV TRAFFICKING AND DC-SIGN-MEDIATED CYTOSKELETON REARRANGEMENT IN THE CONTEXT OF HIV TRANSFER ACROSS DENDRITIC CELLS-T LYMPHOCYTES INFECTIOUS SYNAPSES

Résumé :

La transmission de l’infection par le VIH s’effectue principalement par voie sexuelle et la compréhension des mécanismes liés à ce transfert au niveau des muqueuses est un sujet de recherche primordial afin de développer des outils thérapeutiques à même de prévenir la propagation du VIH. Un rôle majeur des cellules dendritiques muqueuses dans la transmission du VIH aux lymphocytes T a déjà été proposé précédemment.

Ce travail de thèse a permis de caractériser les mécanismes de transfert de l’infection VIH des cellules dendritiques aux lymphocytes T via les synapses infectieuses. Les éléments présentés dans cette thèse apportent des arguments essentiels dans la compréhension des évènements impliqués au niveau de la synapse infectieuse. Nous démontrons ici le rôle de l’activation de la GTPase Cdc42 et des filopodes dans la transmission du VIH aux lymphocytes T. Nous proposons également que l’activation de la GTPase Cdc42 survient suite à la liaison de l’enveloppe du VIH à DC-SIGN et via les kinases SRC. Nos résultats objectivent également que différentes méthodes prévenant la formation de filopodes sur les cellules dendritiques réduisent de façon importante le transfert du VIH aux lymphocytes T.

Ces découvertes ouvrent le champ vers de nouvelles approches thérapeutiques dans la prévention de la transmission du VIH.

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1. ABSTRACT

Around 33.3 million people are estimated to be infected with HIV by the end of year 2010, including 2.5 million children under the age of 15. It is currently estimated that there are between 2.5 and 3 million new infections per year and the rate of new HIV infections has not been dramatically reduced upon highly active anti-retroviral therapy (HAART) implementation. Albeit HIV prevalence among the adult and children population has remained constant during the last years (www.UNAIDS.org), HIV epidemics is still far from over. Additionally, even with a broader access to HAART drugs, there are still near 2 million deaths resulting from AIDS yearly worldwide (www.UNAIDS.org). Currently, the largest number of people infected with HIV can be found in Subsaharrean Africa and this area is also the world region where most of new infections are detected (www.UNAIDS.org). Assuming the great human burden represented by HIV pandemics and the fact that its socio-economical impacts will steadily grow over the next decades, it remains an absolute priority to stop HIV epidemics propagation as soon as possible. In that view, we urgently need to gather accurate understandings about the routes through which HIV infection is transmitted.

Two different subtypes of HIV virus have been described both of them sharing a great level of homology (Campbell-Yesufu et al., 2011). However, most infections are due to HIV-1 and the vast majority of fundamental and clinical research studies have been performed on this subtype, including the work performed during my thesis. HIV transmission primarily occurs through the inclusion of viral particles in various body fluids such as blood, semen, vaginal secretion and cerebrospinal fluid (Flint et al., 2009). Despite the description of some particular HIV acquisitions routes, such as needle-sharing among drug users or blood transfusion, the major way of getting infected with HIV transmission is represented by unprotected sexual intercourse, either via oral, vaginal or anal routes (Flint et al., 2009).

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During these events, HIV viral particles have to cross the oral, vaginal or rectal mucosal barrier in order to reach adequate replicative sites in the new host. The natural protective properties of mucosal epithelia explain why the entrance of HIV to the submucosal tissues is significantly facilitated upon mucosal barrier disruption, as can occur following traumatic sexual intercourse or upon local inflammatory events (Pope and Haase, 2003; Shattock and Moore, 2003). Once they have gained access to the submucosal level, HIV virions can replicate locally before HIV infection spreads systemically throughout the whole body (Haase, 2005). The major clinical impact of HIV infection is the progressive but irremediable decline of CD4⁺ T lymphocytes in infected individuals and the following inability to build up appropriate immune responses against otherwise less-aggressive or innocuous pathogens (Kuritzkes and Walker, 2007).

As they are crossing mucosal barriers, HIV viral particles get in contact with the first line of protection against incoming pathogens. This includes specific immune system cells located in epithelia such as dendritic cells (DC), Langerhans cells (LC), macrophages or lymphocytes.

Dendritic cells have first been described by the 2011 Nobel Prize winner in Physiology or Medicine, Ralph M. Steinman in 1973 (Steinman and Cohn, 1973). Dendritic cells play a pivotal role in mounting efficient immune responses as they greatly contribute to a specific, restricted and efficient response to a wide range of different pathogens including bacteria, mycobacteria, fungi, viruses and parasites (Banchereau and Steinman, 1998). Present in peripheral and lymphoid tissues as immature DC (iDC), DC can potently scan foreign pathogens and are able to induce adequate and specialized immune responses. Detection of an enemy leads to its internalization and degradation into antigens meant for presentation to effector cells of the immune system. DC are thus positioned at the crossroads between innate and adaptive immunity and it is through their innate capabilities of pathogens detection that

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they are able to initiate a correct adaptive immune response (Guermonprez et al., 2002). One of the key features of DC is their ability to switch from an immature state to a mature state of activation. Upon antigen capture, DC enter maturation, an activation process that turns them from highly endocytic and weakly able to mount immune responses immature DC (iDC) into weakly endocytic and potently immunogenic mature DC (mDC). This process of DC maturation is accompanied by their migration to peripheral or proximal lymph nodes where DC get in touch with naive T lymphocytes. The interaction between DC and T lymphocytes at the level of the lymph nodes occurs through close cell-to-cell contacts named immunological synapses. Dynamic contacts consequently induce a substantial and clonal proliferation of T lymphocytes, which is the key point in order to efficiently mount an appropriate adaptive immune response (Banchereau and Steinman, 1998; Lanzavecchia and Sallusto, 2001b).

Despite their innate ability to process pathogens and help the organism clear prom infections, it is also well demonstrated that DC are not fully able to withstand the challenge of HIV infection (Patterson and Knights, 1987). In fact DC are able to degrade the vast majority of incoming HIV virions within the first 24 hrs of infection (Moris et al., 2004; Turville et al., 2004) and are able to present HIV-derived antigens to T lymphocytes, subsequently initiating an immune response that culminates in the generation of HIV specific antibodies initially effective against HIV (Flint et al., 2004). However, the specific status of DC as antigen- presenting cells (APC) helps HIV gain access to its final target cells, T lymphocytes. Whether fully infected or not, DC have been shown to potently boost HIV infection in the context of DC-T lymphocytes co-cultures. Even if the exact level of this enhancement has been subject to debate, it is nevertheless now strongly established that these co-cultures result in higher levels of HIV replication compared to cultures of lone T lymphocytes. (Cameron et al., 1992;

Granelli-Piperno et al., 1998; Pope et al., 1994). The description and characterization of

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infectious (or virological) synapses has helped the scientific community in gaining a better view of how this additional impact of DC-T lymphocytes co-cultures acts on enhanced HIV replication. (McDonald et al., 2003; Piguet and Sattentau, 2004). It is here important to emphasize the structural homology observed between infectious and immunological synapses (McDonald, 2010). Succesive studies have led to the proposal that DC are able to transmit HIV infection to T lymphocytes in a two-phase process (Turville et al., 2004). During the first 24 hrs of exposure to HIV, DC, mostly because of their high ability to capture and endocytose foreign material, are able to bind HIV viral particles through specific cell-surface receptors such as the C-type lectin receptor DC-SIGN. Subsequently, this leads to internalization of intact HIV virions, the majority of them being however consequently degraded (Moris et al., 2004; Turville et al., 2004). Yet, it has been additionally shown that a small fraction of internalized HIV virions can escape proteosomal degradation and that this preserved portion of HIV particles can subsequently efficiently be transferred, in the complete absence of viral replication in DC, to T lymphocytes through DC-T lymphocyte infectious synapse (Arrighi et al., 2004a; Arrighi et al., 2004b; Garcia et al., 2005; Lore et al., 2005; Turville et al., 2004). In addition, a later second step can engage transfer of HIV virions arising from de novo production via viral replication of HIV virions in DC (Garcia et al., 2008; Turville et al., 2004).

Previous data from our laboratory and others have shown that the DC-specific C-type lectin receptor DC-SIGN seems to play an essential role in HIV entry in DC and DC-mediated HIV trans-infection of CD4⁺ T lymphocytes in the lack of viral replication in DC (Arrighi et al., 2004b; Geijtenbeek et al., 2000b; Kwon et al., 2002; Sol-Foulon et al., 2002). The initial steps of HIV journey in DC were also previously described, as a tetraspanin-rich compartment was shown to play a major role in HIV segregation prior to subsequent transfer to T lymphocytes

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across infectious synapses (Garcia et al., 2005). As part of my thesis work, I was initially engaged in the better characterization of the intracellular compartment in which HIV trafficks prior to transfer. Subsequently, the later part of my thesis work was focused in deciphering how HIV hijacks cytoskeletal structures in order to gain access to the infectious synapse and beyond.

Although HIV internalization after capture was shown to be a crucial step prior to viral transfer to T lymphocytes (Arrighi et al., 2004a; Garcia et al., 2005; Kwon et al., 2002;

McDonald et al., 2003), few was known about the nature of the HIV-containing compartment in DC and whether this compartment could also play a role for the long-term transfer of HIV viral particles to CD4⁺ T lymphocytes. As a consequence, we investigated the precise intracellular trafficking of de novo synthesized HIV particles in DC and found that HIV is segregated in a tetraspanin-rich compartment that is enriched in the AP-3 adaptor protein complex involved in HIV Gag trafficking to late endosomal/MVB membranes (Garcia et al., 2008). Furthermore, we were able to show that HIV also trafficks into specific intracellular compartments named immunoamphisomes in DC, where it acts by reducing levels of autophagy via an mTOR signaling pathway in order to increase its subsequent transmission to CD4⁺T lymphocytes (Blanchet et al., 2010).

Having characterized in greater detail the site where HIV resides upon internalization in DC, we aimed at better defining the processes taking places upon DC-SIGN engagement on DC by HIV. Previous works had shown that HIV activates a specific transcriptional program in DC following DC-SIGN binding that culminates in the activation of the guanidine-exchange factor LARG (Hodges et al., 2007). As LARG and other guanidine exchange factors can have profound impacts on Rho GTPases and thus cytoskeleton rearrangement, we postulated that

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HIV-mediated DC-SIGN binding in DC can modulate the cytoskeleton of the cell in order to facilitate viral propagation to T lymphocytes. Interestingly, previous reports had shown that specific cytoskeletal structures could be engaged in order to help HIV viral egress from a infected cell to an uninfected one. This includes filopodia formed between adjacent HIV- infected epithelial cells or T lymphocytes (Sherer et al., 2007; Nobile et al., 2010), nanotubes linking infected T lymphocytes and non-infected T lymphocytes or macrophages (Sowinski et al., 2008; Lamers et al., 2010) or tunneling-nanotubes observed between HIV-infected macrophages (Eugenin et al., 2009).

During my thesis work on this topic, I was able to demonstrate via confocal microscopy techniques and transmission emission microscopy that HIV induces membrane extensions in DC that are similar to filopodia (Nikolic et al., 2011). Moreover, we demonstrated that filopodia activation in DC is induced following binding of HIV^envwith DC-SIGN and subsequent activation of the Rho-GTPase Cdc42 via a Src kinases-dependent pathway (Nikolic et al., 2011). We subsequently characterized further downstream signaling events following HIV engagement of DC-SIGN, showing that Pak1 and Wasp are also activated (Nikolic et al., 2011). We could additionally confirm the major role of Cdc42 in HIV transfer across infectious synapses by the mean of RNAi-mediated silencing of Cdc42 and transfection of dominant-negative or constitutively-active constructs of Cdc42 in DC.

Furthermore, the use of advanced imaging techniques such as tridimensional scanning electron microscopy (3D-SEM) allowed us to definitively assess the role of filopodial membrane extensions in the context of DC-T lymphocyte HIV transfer across infectious synapses (Nikolic et al., 2011).. Moreover, we were also able to determine that Cdc42 and filopodia are of highest importance for HIV fusion in T lymphocytes in the context of DC-T lymphocytes infectious synapses. Finally, we moved to more physiological conditions of HIV

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transfer across infectious synapses, such as lymph nodes-mimicking conditions or analysis of myeloid DC (MyDC) – T lymphocytes co-cultures, and found that Cdc42 was also a key player for efficient HIV transfer in these settings (Nikolic et al., 2011).

Taken together the results presented here further underline the complex relations at play between DC and HIV and might help us understand the complex events taking place in vivo at the level of the mucosae. Further investigations will for sure help improve our knowledge about these early events, paving the way for the generation of potent and efficient vaccines or microbicides (Haase, 2005; Nikolic et al., 2007 ; Nikolic et al., 2010a; Nikolic et al., 2010b).

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2. ABREVIATIONS

AIDS Acquired immunodeficiency syndrome

APC Antigen presenting cell

CDC42 Cell division control protein 42 homolog

CLR C-type lectin receptor

CTL Cytotoxic T lymphocyte

DC Dendritic cell

DC-SIGN DC-specific ICAM-3-grabbing nonintegrin

GALT Gut-associated lymphoid tissue

HAART Highly active anti-retroviral therapy

HIV Human immunodeficiency virus type

iDC Immature dendritic cell

LC Langerhans cell

MDDC Monocyte-derived dendritic cell

MHC-I &-II Major histocompatibility complex class-I and –II

mDC Mature dendritic cell

myDC Myeloid dendritic cell

pDC Plasmacytoid dendritic cell

PRR Pathogen-recognition receptor

R5-HIV-1 CCR5-using HIV-1

X4-HIV-1 CXCR4-using HIV-1

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3. GLOSSARY

Cis-infection pathway

A mode of DC-mediated infection of CD4⁺ T lymphocytes which requires DC infection and de novo synthesis of HIV viral particles in DC for long-term transmission of HIV infection.

Trans-infection pathway

A mode of DC-mediated infection of CD4⁺ T cells which does not require DC infection. DC capture and internalize infectious particles, later transferred to T lymphocytes, in the absence of HIV viral replication in DC.

HIV-1-pulsed DC

DC which, after exposure to HIV, capture and internalize virions but are not infected.

‘Resting’ CD4+ T cells

Refers to in vivo CD4⁺T lymphocytes which exhibit a resting phenotype but however support HIV replication, unlike in vitro resting CD4⁺ T lymphocytes.

Infectious (or virological) synapse

Refers to cell-to-cell contacts through which HIV is transferred from a donor cell (i.e. DC) to a target cell (i.e. CD4⁺ T lymphocyte), whether the donor cell is infected or not (e.g. HIV- pulsed DC).

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4. INTRODUCTION

4a. HIV-1 pathogenesis and its relation to AIDS

Human immunodeficiency virus type 1 (HIV-1) is the etiologic cause of the acquired immunodeficiency syndrome or AIDS (Barre-Sinoussi et al., 1983; Popovic et al., 1984;

Sarngadharan et al., 1984). HIV-1, and its related similar virus HIV-2, is at the origin of one of the major epidemics the human species has had to face in the last centuries. Currently, in 2011 based on the final WHO figures for 2009, it is estimated that around 33.4 million people are infected worldwide with HIV. More importantly, there are still about 2.6 million new infection cases occurring every year, even if this figure has decreased of about 25% since 1999 when the highest overall incidence rate for HIV infection was recorded. Despite a simpler access to active highly active anti-retroviral therapy (HAART), as much as 1.8 million people die every year worldwide from AIDS (www.UNAIDS.org). Even though these figures may seem encouraging when compared with those of the beginning of the last decade, there still represent a massive human burden. Especially, some Sub-saharrean countries still demonstrate prevalence rates of about 40 to 50% amongst the total population, indicating that almost half of their population will have to deal with AIDS-related co-morbidities in a near future unless they gain access to anti-retroviral therapy. The harshness and the implications of HIV epidemics in terms of human and economical costs, remain major issues, particularly in developing countries (Fig.1) (Piot et al., 2001).

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Figure 1. HIV prevalence throughout the world in 2008. (source: WHO/UNAIDS)

As it will be explained in more detail further in this text, HIV cell tropism is determined by the sequential use of CD4 and one additional specific co-receptor (either CXCR4 or CCR5) during entry into target cells. As a consequence, CD4⁺ T helper lymphocytes, macrophages (which express CD4) and some CD4⁺ dendritic cells (DC) (also expressing CD4) subsets are the main targets of HIV (Freed and Martin, 2007). The chief clinical outcome of HIV infection is the progressive and continuous depletion of CD4⁺helper T lymphocytes in infected individuals (Fig. 2). This consequently leads to a progressive inability of the immune system to generate correct immune responses towards foreign pathogens (Kuritzkes and Walker, 2007).

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Figure 2. Classical clinical course of HIV infection. (source: NIAID/NIH)

Progression towards AIDS can be divided into three different stages (Flint et al., 2009).

During the first (acute) phase, HIV replicates to very high levels in activated CD4⁺ T lymphocytes, inducing generally only moderate flu-like symptoms in infected individuals (Kahn and Walker, 1998). However, as a key feature of its pathogenesis, HIV also infects resting memory CD4⁺ T lymphocytes in mucosal lymphoid tissues (MALT), notably the gut- associated lymphoid tissue (GALT), leading to a massive and irreversible depletion in T lymphocytes in these tissues (Brenchley et al., 2004; Mehandru et al., 2004). Nevertheless, HIV replication is usually reduced to quite low levels during the first weeks or months of infection. This can potentially be explained by the cell-mediated immune response, as the number of circulating activated CD8⁺ T lymphocytes (or cytotoxic T lymphocytes (CTL)) is known to increase before the occurrence of specific neutralizing antibodies. Seroconversion from an HIV-negative to HIV-positive status is typically used by physicians as the best

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marker of HIV infection (Gaines et al., 1987; Schmitz et al., 1999). Interestingly the initial control of viral replication results in CD4⁺ T lymphocytes global counts rapidly returning to levels close to the initial values observed prior to infection (Fig. 2). At the end of this first phase the residual levels of HIV viremia, the so-named virological set point, can be used as an appropriate indicator of disease progression, with high viremia usually linked to fast progression to AIDS. Clinically, this stage of HIV infection is generally characterized by few clinical symptoms; it is therefore frequently also named as the asymptomatic phase of HIV infection. During this period of time that can last until years in untreated patients, CD4⁺ T lymphocytes classically decrease in number while viral replication still actively persists in lymph nodes (Fig. 2). The combination of the direct cytopathic effect of HIV on CD4⁺ T lymphocytes and the simultaneous CD8⁺ T lymphocyte-mediated killing of infected lymphocytes are the major reasons explaining the gradual drop in CD4⁺ T lymphocytes over time. Additionally, it has been demonstrated that HIV preferentially infects HIV-specific memory CD4⁺ T lymphocytes, thus destroying the most susecptible cells that might mount efficient responses to it (Douek et al., 2002). During this second, asymptomatic, phase opportunistic infections and malignancies related to AIDS are generally rare issues in HIV- infected patients as long as CD4⁺ T lymphocytes counts remain above 400 cells/μl. These AIDS-related conditions usually begin to appear with CD4⁺ T lymphocytes below these values. Indeed, their incidence starts to dramatically increase when CD4⁺ T lymphocytes reach values under 200 cells/μl. Accumulating scientific proofs show that, besides viral immune escape, HIV-mediated killing of CD4⁺ T helper cells and the architectural modification of lymph nodes greatly favor the occurrence of AIDS-related opportunistic infections and malignancies (Kuritzkes and Walker, 2007; Stevenson, 2003). Virus-specific CD4⁺ T helper lymphocytes, that orchestrate both cellular and humoral immune responses, should classically assist CD8⁺ T lymphocytes in generating effective CTL-response that could

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contain chronic HIV infection (Day and Walker, 2003; Wherry et al., 2003). However, loss of HIV-specific CD4⁺ T helper lymphocytes in the context of HIV infection renders CD8⁺ T lymphocytes functionally ineffective (Douek et al., 2002; Flint et al., 2009). The emergence of potentially effective treatments over the two last decades has led to the appearance of potent combinatory antiretroviral therapies. Since its introduction in the mid-90s, highly active antiretroviral therapy (HAART) has radically and strongly reduced AIDS-related mortality and morbidity. (Egger et al., 2002; Ickovics et al., 2002; Parker et al., 1991).

Patients under HAART generally exhibit HIV RNA levels around the limit of viral RNA detection (50 copies of HIV RNA per ml of plasma) (Flint et al., 2009). However, HAART does not totally clear HIV infection and viral replication can still arise in specific

“sanctuaries”, such as resting CD4⁺T lymphocytes, macrophages and most likely also DC at very low levels. Basically known as cellular reservoirs, these viral sanctuaries allow HIV to escape HAART and host immune responses (Finzi and Siliciano, 1998; Stevenson, 2003).

Amongst other factors, poor compliance to therapy is often seen as the main reason standing behind HAART failure (Ickovics et al., 2002). Emergence of multiple drug-resistant genotypes of HIV additionally complicates treatment in these patients. Although vaccinal approaches against HIV have improved over the last years (Padian et al., 2011), it remains of greatest importance that we keep on dissecting HIV viral cycle and pathogenic behavior in order to generate new potent therapeutic targets. New insights into the precise mechanisms taking place during HIV mucosal transfer will hopefully lead to the generation of efficient and economically affordable methods to both prevent and cure HIV infection.

4b. Classification of HIV-1

HIV is classified as a member of the Retroviridae family and is therefore characterized as an enveloped RNA virus using a specific replication cycle. The particularity of retroviruses is

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that their genomic information switches from RNA to DNA after cell entry but prior to viral DNA integration into the host genome (Goff, 2007). Phylogenetic analyses have placed HIV- 1 and HIV-2 in the lentivirus genus, beside other mammalian viruses such as bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), feline immunodeficiency virus (FIV) and simian immunodeficiency virus (SIV). Lentiviruses are known to cause slow, chronic diseases in their respective hosts by targeting cells of the hematopoietic lineage, specifically lymphocytes and macrophages (Freed and Martin, 2007). Lentiviruses have a more complicated genomic organization than the so-called simple retroviruses, such as mouse mammary tumor virus (MMTV) or murine leukemia virus (MLV) (Goff, 2007). As a consequence, additionally to the usual structural and enzymatic proteins common to all retroviruses, HIV encodes six specific auxiliary proteins crucial to adapt the cellular and systemic environment of the host (Fig. 3) (Freed and Martin, 2007). Emergence of HIV in the human population was shown to have occured through interspecies transmissions of SIV from chimpanzees (Gurtler et al., 1994; Peeters et al., 1997; Simon et al., 1998). Assuming a globally constant rate of evolution, introduction of HIV into the human population has been evaluated to have occured around 1930 (Korber et al., 2000). In fact this recent outbreak in the human species implies that HIV and humans have therefore just started to co-evolve. This recent onset of co- evolution could explain the high lethality of HIV in its “new” host (Telenti, 2005).

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Figure 3. Structure of the HIV-1 virion and the viral genome. Schematic representation of a HIV-1 virion. HIV-1 genomic organization and the resulting three polyproteins. Env:

envelope, p17: matrix, p24: capsid, p7: nucleocapsid, RT pol: reverse transcriptase, IN:

integrase, PR: protease.

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4c. HIV-1 replication cycle

HIV virion structure

Mature HIV virions are roughly spherical particles 120 nm in diameter surrounded by a lipid bi-layer of cellular origin covered with trimers of the viral envelope glycoprotein (Env) (Fig.

3) (Freed and Martin, 2007). The gp160 Env protein precursor is cleaved into gp41 and gp120 subunits which together represent the Env viral protein of HIV (Freed and Martin, 2007).

Under the lipid bilayer, a web of HIV matrix protein (p17/MA) is found to be clustered around electron-dense cone-shaped core localized at the center of mature particles. The external layer of the viral core is subsequently characterized by an agglomeration of viral capsid protein (p24/CA) while its internal part is formed by two copies of genomic RNA closely bound to viral nucleocapsid proteins (p7/NC). The genomic HIV material linked to p7/NC is associated within the core with the viral reverse transcriptase (p66-p51/RT) and the viral integrase (p32/IN) (Fig. 3).

Viral entry

The first step in HIV replication cycle is represented by the internalization of mature viral particles at the surface of target cells (Fig. 4). Binding to the CD4 receptor by the gp120 subunit of Env is essential for HIV infectivity and subsequently also defines its cell-tropism (Freed and Martin, 2007). CD4 binding to the cell-surface of target cells is moreover the key phenomenon engaging Env major conformational changes that culminate in virion fusion at the target cell plasma membrane and cell entry. (Deng et al., 1996; Feng et al., 1996).

Subsequent interactions with the chemokine receptors CXCR4 or CCR5 enhance the fusogenic capacity of gp41 and thus HIV virion fusion with the target cell(Doms and Trono, 2000; Sattentau and Moore, 1991). HIV strains are usually classified as R5-, X4- or dual

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R5/X4-viruses according to the co-receptor engaged (CCR5, CXCR4, or both, respectively) (Berger et al., 1998; Doms and Trono, 2000). R5-viruses are the principal strain of HIV found in recently infected individuals (Zhu et al., 1993). Later on during HIV infection is it usual to observe a gradual increase in X4-tropic virus and it has been also demonstrated that the increase in X4-tropic virus can be an indicator of progression to AIDS. (Connor et al., 1993).

Figure 4. Classical representation ofHIV replication cycle.

Reverse transcription and integration

After fusion and entry, HIV enters a process of uncoating that releases viral cores into the cytoplasm of the target cell (Fig. 4). The remaining of HIV virion at that point is composed from the genomic RNA, the viral RT and IN, but also from other viral and cellular components (Greene and Peterlin, 2002). Reverse transcription is starting at that point with the initial synthesis of a minus-strand DNA from the genomic RNA. At the same time as

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DNA synthesis towards the 5’ end of the genome occurs, does the RNase H activity of RT digests the RNA part of the newly formed DNA/RNA complex, releasing the minus-strand strong-stop DNA intermediate. Final to complex processes of strand transfers is it possible to obtain a double stranded HIV viral DNA flanked with long terminal repeats at both ends. The end product of this process is the HIV pre-integration complex (PIC) that is formed of double- stranded viral cDNA, the viral IN, RT, MA, Vpr and NC proteins along with other cellular factors. The PIC’s role is to target HIV genomic information to the host cell nucleus (Freed and Martin, 2007; Greene and Peterlin, 2002; Miller et al., 1997). A key property of lentiviruses is their ability to infect non-dividing cells, such as resting T lymphocytes or dendritic cells. A limiting step for the intra-nuclear entry of the PIC of HIV is therefore represented by the step of active viral nuclear entry. HIV matrix protein (MA) bears a nuclear location signal (NLS) recognized by the components of the nuclear-import pathway (Bukrinsky et al., 1993). Even though they lack canonical NLS, IN and Vpr could also play a role in active PIC nuclear transport (Gallay et al., 1997; Heinzinger et al., 1994). The exact contributions of each viral factor in this process is however still subject to debate and remains to be further specified (Freed and Martin, 2007). HIV genomic material (in a DNA form) integration is then catalyzed by HIV integrase (IN). After removal of two nucleotides from each viral LTR, IN induces the integration of the free viral cDNA into the host chromosome as an integrated provirus. Next, the host cellular DNA repair mechanism fills the gaps created during this process, therefore terminating integration (Greene and Peterlin, 2002).

Viral proteins synthesis

HIV proviruses can integrate at different chromosomal locations throughout the genome of infected T lymphocytes in an either latent or transcriptionally active manner. In this transcriptionnally active situation, HIV provirus transcription is performed by the host cell

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RNA polymerase II (RNApol II). In this circumstance, the 5’ LTR serves as a transcriptional unit and provides a true platform that recruits the RNApol II via classical promoter elements such as the TATA box or Sp1 sites (Greene and Peterlin, 2002). Upstream sequences serve as transcriptional enhancers and can engage the nuclear factor κB (NF-κB) and the nuclear factor of activated T cells (NFAT), which subsequently induce transcription. HIV accessory viral protein Tat plays a crucial role at this step as it, in association with cyclin T1, binds the transactivation response (TAR) element, an RNA stem loop present on aborted viral RNAs, and further recruits the cellular cyclin-dependent kinase 9 (Cdk9) in order to help RNApol II elongate viral DNA (Wei et al., 1998). Before export out of the nucleus to the cytoplasm, postranscriptional modifications on viral RNA are performed. However, distinct to eukaryotic mRNA, some HIV mRNA is differentially exported to the cytoplasm either unspliced (genomic RNA), partially spliced (Env, Vif and Vpr mRNA) or multiply spliced (Nef, Tat and Rev mRNA). Incomplete splicing originates from the incorporation into HIV transcripts of poor splice-donor and acceptor sites combined with the action of the regulator of expression of viral proteins (Rev). Rev accessory protein is an RNA-binding protein that recognizes the Rev-responsive element (RRE) present in env. This next activates the export of unspliced or partially spliced viral mRNA through interplay with Crm1, a member of the importin-β family involved in nuclear export (Freed and Martin, 2007). Larger amounts of Rev protein finally massively favor export of incompletely spliced viral transcripts, encoding the structural, enzymatic and most of the accessory viral proteins (Cullen, 1998).

HIV viral assembly

HIV genome-length unspliced viral transcripts give rise to the viral polyprotein precursors Gag and Gag-Pol which direct assembly and budding of newly synthesized virions (Fig. 4).

The different domains of Gag and Gag-Pol play significant roles in the various steps leading

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to HIV budding from infected cells. Gag and Gag-Pol polyproteins basically multimerize through direct protein-protein contacts linking their respective CA domains (Freed, 1998).

This multimere subsequently recruits two copies of the viral genome through the interaction of their NC domain with the ψ site, a specific viral RNA packaging structure present just downstream of the 5’ LTR (Freed, 1998). These Gag-RNA complexes are subsequently

targeted to the inner leaflet of the plasma membrane via the myristoilated moiety found at the N-terminal part of the MA domain (Gottlinger et al., 1989). Association within the plasma membrane is then observed in cholesterol- and glycolipid-enriched membrane domains, the lipid rafts (Ono and Freed, 2005). Additionnaly, budding through these plasma membrane domains enhances HIV release and its fusogenic capacity (Campbell et al., 2001). The Env polyprotein gp160 is synthesized in the endoplasmic reticulum (ER), cleaved in the Golgi apparatus and sent to the cell surface through the classical secretory pathway (Fig. 4). A subsequent step is represented by the incorporation of the mature subunits of Env, gp120 and gp41 at the moment of viral budding. This step is mediated through direct interactions between Env and the MA domain of Gag and Gag-Pol multimers. At the same time, HIV virions can be released with several cellular surface proteins, like human leukocyte antigen (HLA) -I and –II or intercellular adhesion molecule-1 (ICAM-1) (Freed and Martin, 2007).

Accumulation of Gag and Gag-Pol multimers at the plasma membrane self-induces plasma membrane curvature and promotes the formation of immature HIV particles that are tethered to the membrane via the cellular protein tetherin which action is counteracted by the viral accessory protein viral protein U (Vpu) for HIV-1 (Neil SJ et al., 2008) or Env for HIV-2 (Hauser H et al., 2010). The final step in HIV budding involves release of synthesized virions from the plasma membrane. This process is dependent on the last domain of the Gag precursor, the p6 domain. Through its PTAP motif, p6 interacts with the tumor suppressor gene 1 (Tsg101), a member of the endosomal sorting complex required for transport-I

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(ESCRT-I). This binding allows HIV divert to its own benefit the cellular machinery usually involved in budding of vesicles into the lumen of the late endosome/multivesicular body (MVB) (Garrus et al., 2001; Katzmann et al., 2001). Besides these budding properties described in classical cell types (T lymphocytes) some HIV-permissive cell types such as macrophages show assembly and budding of newly synthesized virions in intracellular compartments (Pelchen-Matthews et al., 2003). Several recent studies in macrophages would tend to combine these two observations as plasma membrane invaginations seem to be the source of the HIV-containing compartment in these cells (Deneka et al., 2007; Jouve et al., 2007; Welsch et al., 2007).

Accessory proteins

In addition to its structural, enzymatic and regulatory proteins, HIV genome encodes 4 different accessory proteins (Fig. 3). We will here discuss the currently established functions of these 4 proteins.

- Nef: Nef was first described as a factor decreasing viral replication in infected cells and was thus named as “viral negative factor,” which was later found to be quite inaccurate do to its real function. Basically, HIV Nef has been shown to downregulate several cell-surface molecules such as CD4, MHC-I and MHC-II (Piguet et al.,1998; Schwartz et al., 1996;

Stumptner-Cuvelette et al., 2001). In the case of CD4 and MHC-I, Nef directly anchors the cytoplasmic domain of this protein and targets it towards lysosomal degradation. In addition, Nef was also shown to down-modulate the expression of MHC-I as well as MHC-II. The reduced expression of CD4 on the cell surface avoids overinfection and favors HIV release.

Low cell-surface levels of MHC-I represent a clear advantage in avoiding CTL-mediated killing of HIV-infected cells (Collins et al., 1998). The associated low MHC-II-restricted

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peptide presentation additionally impairs anti-HIV immune responses by avoiding the full activation of HIV-specific CD4⁺T lymphocytes (Freed and Martin, 2007). Furthermore, Nef interferes with cellular signal transduction pathways and modulates T-lymphocyte activation, with the consequence of favoring HIV replication (Fenard et al., 2005; Krautkramer et al., 2004). Interestingly in the context of this thesis, Nef was also recently shown to induce filopodia in T lymphocytes (Nobile et al., 2010).

- Vif: The viral infectivity factor (Vif), crucial for HIV replication, is produced to high levels in infected cells. The role of Vif is mainly related to the cellular restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) (Harris et al., 2003). APOBEC3G is a cellular DNA cytidine deaminase that induces G-to-A hypermutations of the viral genome of Vif-deficient HIV virions (Lecossier et al., 2003;

Mangeat et al., 2003; Zhang et al., 2003). Additional studies have demonstrated that Vif counteracts the deaminating activity of APOBEC3G by targeting this restriction factor for degradation (Mehle et al., 2004; Yu et al., 2003).

- Vpr: Vpr is significantly incorporated into virions mostly through its interaction with the p6 domain of Gag (Kondo and Gottlinger, 1996; Lu et al., 1995; Yuan et al., 1990). Vpr was shown to have a substantial role in non-dividing monocyte-derived macrophages (MDM) (Freed and Martin, 2007). Data from Vpr-deficient HIV strains isolated from long term non- progressors point toward a significant role for Vpr in vivo (Lang et al.,1993; Somasundaran et al., 2002). In addition, the presence of Vpr in the PIC of HIV and in the nucleus of infected cells strongly suggests a role for this protein in the nuclear import of viral components (Heinzinger et al., 1994; Lu et al., 1993). Vpr has also been shown to moderately induce gene expression from the HIV LTR and to induce cell-cycle arrest in the G2 phase (Cohen et al.,

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1990; Poon et al., 1998). As HIV transcription mainly occurs during the G2 phase, the cell- cycle arrest function of Vpr therefore probably provides a favorable nuclear environment to HIV (Goh et al., 1998).

- Vpu: Unique to HIV-1, the viral protein U (Vpu) is an integral membrane protein involved in CD4 degradation and in the enhancement of viral particle release. Proteasomal degradation of CD4 occurs after Vpu binding to CD4 and to the cellular factor targeting proteins to the proteasome, β-TrCP (Freed and Martin, 2007), in the endoplasmic reticulum. Degradation of CD4 releases the Env precursor gp160 and allows Env to proceed to the cell surface, thus increasing HIV-1 particles infectivity (Willey et al., 1992a; Willey et al., 1992b). In parallel, Vpu has been shown to increase HIV-1 particle release by counteracting the effects of the cellular inhibitor of virus release named tetherin (Neil et al., 2008; Van Damme et al., 2008).

4d. Dendritic cells

The main route of HIV acquisition worldwide results from unprotected sexual intercourse (Flint et al., 2004). Following this pathway, HIV enters the host across mucosal surfaces before spreading throughout the organism. Amongst the variety of cells present at the mucosal level, dendritic cells (DC) represent the first line of defense against incoming virions.

However, HIV has managed to overcome the barrier represented by dendritic cells and has in fact turned DC into one of its most powerful allies.

DC: a cannonical antigen presenting cell

DC represent a varied cellular population that plays a central role in acquired immunity.

Along with other antigen presenting cells (APC), mainly macrophages and B lymphocytes, they are the key organizers of proper, controlled efficient T cell responses (Banchereau and

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Steinman, 1998). APC function through the presentation on their cell surface of foreign antigens that are recognized by T lymphocytes. This process involves antigen uptake, its lysosomal degradation and its loading on major histocompatibility complex (MHC) class I and II molecules. This subsequently leads to processed antigens presentation to T lymphocytes. All APC do not function equally for this task however. For example, B lymphocytes only weakly endocytose foreign material. They are however fully able to present antigens to T lymphocytes. B lymphocytes-antigen presentation is nonetheless essential as it participates to the onset of humoral immune response that culminates with the secretion of specific antibodies by plasma cells (Trombetta and Mellman, 2005). On the other side, macrophages exhibit high levels of endocytosis and relatively low levels of MHC-I and -II molecules on their cell surface which makes them quite weak antigen presenters. It rather seems that the high endocytic capacity of macrophages is mainly aimed towards pathogen clearance (Trombetta and Mellman, 2005). In counterpart, DC, when compared with B lymphocytes and macrophages seem to be specialized for professional antigen presentation.

DC are present in all peripheral tissues where they directly scan the environment and act as gate-keepers for the immune system. DC also constitutively circulate through peripheral tissues, the lymphatic system and the secondary lymphoid organs, reaching sites enriched in T lymphocytes. Constant renewal of DC in peripheral tissues is assured through perpetual migration of blood DC-precursors originating from the bone marrow to peripheral tissues (Banchereau and Steinman, 1998). DC are able to activate naïve T lymphocytes and subsequently induce their proliferation and differentiation in order to launch adaptive immune responses (Banchereau and Steinman, 1998; Lanzavecchia and Sallusto, 2001b). DC establish specific cell-to-cell contacts with T lymphocytes. These close inter-cellular contacts are called immunological synapses (Bromley et al., 2001). At the level of immunological synpases, the T lymphocytes receptor (TCR) recognizes antigenic peptides-MHC (p-MHC) complexes.

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Multiple TCR triggering by p-MHC complexes induces signaling events in the T lymphocyte.

These signaling events subsequently determine the fate of the T lymphocyte (Lanzavecchia and Sallusto, 2001b). The intensity of the signaling cascade induced in T lymphocytes depends on the amounts of p-MHC complexes present in immunological synapses. Ensuing engagement of DC co-stimulatory molecules (CD86 and CD80) by the T lymphocyte co- stimulatory receptor (CD28) additionally amplifies the amount of the initial signal. The temporal length of the DC-T lymphocyte interaction also has a profound impact on the level of signaling in T lymphocytes (Lanzavecchia and Sallusto, 2001a). In parallel to robust T cell activation, DC also polarize T lymphocyte development by inducing peripheral T lymphocyte tolerance to tissue-specific antigens (Lanzavecchia and Sallusto, 2001b).

Dendritic cell functions

DC are active in antigen uptake through the endocytosis of whole pathogens, infected and dead cells or their derived products. Three different mechanisms have been described for pathogen uptake by DC: macropinocytosis, phagocytosis and receptor-mediated endocytosis (Banchereau and Steinman, 1998). DC are able to collect large amounts of extra-cellular fluids for antigens through macropinocytosis. Although a rather unspecific route, this actin- based process allows DC to screen their environment for soluble antigens derived from infection (Guermonprez et al., 2002). Phagocytosis, a receptor-mediated actin-dependent process, allows DC to engulf large intact microbes (i.e. bacteria) or apoptotic cells. Through the expression of a great variety of cells surface receptors, DC are also able to internalize antigens in a more specific manner. Receptor-mediated endocytosis makes antigen uptake very efficient by a concentration effect, making only reduced amounts of antigens sufficient to mount an immune response (Banchereau and Steinman, 1998). Several receptors have been involved in antigen uptake. This includes Fc receptors, scavenger receptors and the C-type

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lectin family of receptors (CLR). CLR bind their ligands through a common carbohydrate recognition domain (CRD) in a calcium-dependent manner. DC express a great number of these lectins like the macrophage-mannose receptor (MMR), DEC-205, the DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) and Langerin (Guermonprez et al., 2002).

Endocytosis of CLR is entirely dependent on their intracellular internalization motifs and may provide differential alternative routes to respond to antigens. Many pathogens target these receptors and seem to obstruct their function, including viruses, bacteria, parasites or fungi (van Kooyk et al., 2004). DC-SIGN, which is only and specifically expressed on DC, was shown to bind HIV and favor infection of co-cultured T lymphocytes, a process termed trans- infection (Geijtenbeek et al., 2000b). DC-SIGN/HIV interactions, which are on the main focuses of my thesis work, will be described with further details in the corresponding section of this manuscript.

Antigen presentation in DC

In DC, like in all APC, antigens are processed into peptides and presented associated with MHC molecules. The resulting p-MHC complexes subsequently serve as recognition signals for the priming of CD8⁺and CD4⁺ T lymphocyte responses. Most peptides loaded on MHC-I are assumed to have an endogenous origin. In this case, antigen degradation involves the proteasome and hence implicates ubiquitination of proteins present in the cytosol. Then, via the peptide transporter TAP, peptides cross the ER membrane and are loaded on MHC-I molecules before p-MHC-I complexes are sent to the cell surface via the Golgi apparatus. In the context of a viral infection, this antigen-presenting pathway allows the mounting of virus- specific CTL-responses only if DC are effectively infected. This is nevertheless not always the case and DC have to use an alternative route named cross-presentation. This process occurs mostly in a TAP-dependent manner and implies that exogenous antigens reach the

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cytosol from the endocytic pathway. Cross-presentation in a TAP-independent mode has also been described (Trombetta and Mellman, 2005). Concerning MHC-II molecules, antigen loading arises at different intracellular sites. MHC-II components traffic through the ER and the Golgi apparatus before being transported to the endocytic pathway where antigenic peptides are efficiently loaded. p-MHC-II complexes are then sent to the cell surface where they can get in contact with T lymphocytes. Endogenous plasma membrane and endosomal proteins as well as internalized exogenous proteins are the source of the antigenic peptides presented on MHC-II molecules (Guermonprez et al., 2002; Trombetta and Mellman, 2005).

Antigens present in the cytosol or in the nucleus can also be found associated with MHC-II molecules. Transfer of cytosolic antigens to the endocytic pathway seems to occur at the levels of lysosomes by a process called autophagy. Autophagy allows the cell to digest parts of its cytosol in order to recycle nutrients or destrox cytoplasmic components or pathogens. In addition, autophagy was shown to link cytoplasmic antigens with MHC-II presentation (Levine and Deretic, 2007). DC express another type of MHC molecules involved mainly in glycolipids presentation to T cells. These molecules, the CD1 proteins, associate with antigens in the endosomal system where CD1 molecules are internalized from the cell surface. Upon presentation on DC cell surface, peptide-CD1 complexes notably activate CD8⁺ T cytotoxic lymphocytes and natural killer T (NKT) cells (Barral and Brenner, 2007; Trombetta and Mellman, 2005).

DC maturation process

A specific developmental process intrinsic to DC segregates them in two groups with distinct functional and phenotypical features: immature (iDC) and mature DC (mDC) (Fig. 5) (Guermonprez et al., 2002). Present in peripheral and lymphoid tissues, iDC are capable of very efficient internalization of antigens while remaining poor T lymphocytes activators. This

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is partly because of low levels of surface MHC-I/II molecules and co-stimulatory proteins CD86 and CD80, both essential for T lymphocyte activation (Fig. 5) (Mellman and Steinman, 2001). MHC-II molecules are significantly expressed in iDC but are generally retained within lysosomal compartments where antigens accumulate after uptake. Although antigens and MHC-II molecules are to be found at the same intracellular location, iDC are inefficient in forming p-MHC-II complexes (Mellman and Steinman, 2001). DC also detect pathogens through their direct recognition via pathogen-associated molecular patterns (PAMPS) or through the indirect sensing of inflammatory cytokines generated by the infection (Guermonprez et al., 2002). Both signals induce DC maturation, thus increasing antigen uptake, antigen processing and presentation. Maturation is therefore a link between innate and adaptive immune responses (Mellman and Steinman, 2001). Besides receptors for antigen uptake, DC exhibit a variety of surface receptors that serve as sensors of pathogens. Pattern- recognition receptors (PRR), such as the Toll-like receptor (TLR) family, allow DC to detect a broad spectrum of pathogenic compounds such as lipopolysaccharide (LPS) (TLR4), unmethylated CpG DNA compounds (TLR9) or single-stranded RNA (TLR7) (Iwasaki and Medzhitov, 2004; Miyake, 2007). DC also sense infection through the effect of inflammatory mediators (TNF-α, IL-1β) on cytokines receptors. Ligand-receptor interactions induce a signaling cascade initiating maturation through the activation of NF-κB, a trait of mature DC (Guermonprez et al., 2002). Such a wide variety of surface receptors suggests that DC can react differentially to diverse stimuli thus adapting their maturation status to the pathogenic situation (Mellman and Steinman, 2001).

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Figure 5. DC maturation. Maturation is a developmental program that modifies the location and the function of DC. Adapted from (Hackstein and Thomson, 2004).

Activation of maturation modifies many aspects of DC biology. As iDC turn into mDC, endocytic uptake of antigens via downregulation of macropinocytosis and phagocytosis and subsequent reduction of antigen receptor surface expression occur. In parallel, MHC-II molecules relocalize from their intracellular lysosomal compartments to the plasma membrane. Concomitantly, maturing DC upregulate antigen proteolysis enabling mDC to efficiently generate p-MHC-II complexes (Delamarre et al., 2005; Trombetta et al., 2003). In addition to increased cell surface levels of co-stimulatory proteins, MHC-I and T lymphocyte adhesion molecules, p-MHC-II-harboring mDC turn into highly potent inducers of T lymphocyte stimulation (Guermonprez et al., 2002; Lanzavecchia and Sallusto, 2001b;

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Mellman and Steinman, 2001). In the context of maturation, DC modify their cellular morphology and start extending long membrane folds known as dendrites. Maturation also induces DC to migrate from peripheral tissues to T lymphocyte-enriched zones of lymph nodes, where T lymphocyte activation will take place. Direct DC-T lymphocytes interactions within lymph nodes are an essential step for final DC maturation. Although these observations help create a comprehensive model, the in vivo reality of antigen presentation is probably more complex. The paradigm implies that DC capturing antigen in the periphery present it to T lymphocytes in lymph nodes. This might not be the case and antigen-loaded DC might first pass antigens to other DC that in turn will present it to T lymphocytes.

Dendritic cells subtypes

Alternate from their maturation status, DC can be classified according to their progenitors, their surface markers or their tissue distribution. CD34⁺ hematopoietic stem cells originate from the bone marrow and give rise to all blood cells including DC in a constitutive, antigen- independent manner (Banchereau and Steinman, 1998; Liu, 2001). Then, CD34⁺ stem cells differentiate into common lymphoid progenitors (CLP) and common myeloid progenitors (CMP) which give rise to the lymphoid or myeloid DC lineages, respectively. CD11c^- CD123⁺plasmacytoid DC (pDC) and CD11c⁺CD123^- myeloid DC (myDC) are both found in the blood and have been considered as “precursors” of tissue DC (Grouard et al., 1997). pDC play a central role in innate immune responses to viral infection by secreting large amounts of class I interferons (INF) before becoming antigen presenting DC in the T lymphocytes zones of lymph nodes (Shortman and Liu, 2002). Blood myDC rather increase interleukin (IL)-12 secretion that can enhance both innate and acquired immunity (Banchereau and Steinman, 1998). Different subtypes of blood myDC migrate into multiple tissues, such as the skin or genital mucosa, and differentiate into two distinct DC populations: the epidermal Langerhans

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cells (LC) and dermal or interstitial DC (Caux et al., 1996; Ito et al., 1999; Liu, 2001). Under inflammatory conditions, blood monocytes are recruited to peripheral tissues and can in turn differentiate into myDC very similar to interstitial DC (Shortman and Naik, 2007). Because of the paucity of DC in vivo, in vitro generation of functional DC has proven essential for analysis of DC function. A major improvement has arisen with the establishment of in vitro culture systems that allow the induction of DC from precursors such as CD34⁺ cells obtained from umbilical cord blood and normal blood monocytes (Arrighi et al., 1999; Romani et al., 1996; Sallusto and Lanzavecchia, 1994; Santiago-Schwarz et al., 1992). Most studies investigating the immunological or virological functions of DC have used monocyte-derived DC (MDDC) as substitute myDC, due to their similar characteristics (Steinman et al., 2003).

4e. HIV/DC interactions

As explained earlier in this introduction, HIV infection is mainly acquired through sexual contacts. In model systems for sexual transmission, resident dermal myDC and LC are supposed to play a central role in the early stages of HIV propagation (Piguet and Steinman, 2007; Pope and Haase, 2003; Shattock and Moore, 2003). In the first moments after contact between mucosa and HIV, very small quantities of HIV must cross genital or rectal mucosal surfaces in order to reach sites of replication troughout the organism. Mucosal surfaces can be divided into single-layered and pluristratified epithelia. The first group is represented by mucosal tissues of the small and large intestine, the pseudostratified epithelia of the respiratory tract, the rectum and the upper female reproductive tract. The pluristratified mucosal surfaces of the oral cavity, the vagina, the ectocervix, the inner foreskin and penile glans are composed of multiple layers of squamous epithelium, which role is to provide a physical barrier to invading pathogens (Iwasaki, 2007). Several mechanisms of mucosal barrier crossing have been described for HIV during viral sexual transmission (Pope and

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Haase, 2003; Shattock and Moore, 2003). Either cell-associated or free HIV virions have been shown to pass from the apical to the serosal pole of epithelial cells by transcytosis (Fig.

10b) (Alfsen et al., 2005; Bomsel, 1997; Bomsel and Alfsen, 2003). This process implies the endocytosis and vesicular transport of viral particles in epithelial cells and does not require viral replication following infection of the epithelial cell. Intraepithelial LC can also capture HIV particles in the lumen of mucosal epithelia and subsequently transfer HIV virions to neighboring permissive cells present in the submucosa via extending cellular processes in between epithelial cells (Rescigno et al., 2001). Finally epithelial cells themselves can get infected in a CD4-independent/CXCR4-dependent manner (Delezay et al., 1997). Mucosal epithelia are indeed quite a difficult hurdle to overcome for incoming viruses such as HIV.

However, breaches in the mucosal barrier caused by physical abrasion or trauma following intercourse and even mucosal inflammation are quite frequent in vivo. These situations certainly help HIV cross the mucosal barrier and reach final replicative sites (Pope and Haase, 2003; Shattock and Moore, 2003). The first in vitro studies on DC/HIV interactions demonstrated an interesting feature that is at the origin of all the further studies involved in the comprehension of DC/HIV interactions. Blood DC, when shortly (30-120 min) pulsed with HIV are able to dramatically increase viral replication in DC-T lymphocytes co-cultures (Cameron et al., 1992). It was in fact shown that DC transfer HIV infection to CD4⁺ T lymphocytes in DC-T cell clusters, where a rapid and productive infection takes place (Cameron et al., 1992). Because the resulting level of CD4⁺ T lymphocyte infection is higher than the infection level of infected CD4⁺ T cells alone, this DC characteristic was named trans-enhancement of infectivity. These findings were later confirmed in MDDC as well as LC and dermal DC emigrated from skin explants (Granelli-Piperno et al., 1998; Pope et al., 1994). In the later experimental conditions designed to modelize HIV transmission at mucosal surfaces, infection of the LC/DC themselves is required for an efficient HIV transmission to

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CD4⁺ T lymphocytes, in contrast with blood DC which do not need full viral replication to transfer infection (Cameron et al., 1994; Pope et al., 1995).

HIV cis-infection of DC

Other experimental works have clearly shown that HIV can infect LC and other DC subtypes, both in vitro or in vivo (Berger et al., 1992; Granelli-Piperno et al., 1998; Patterson et al., 2001; Pion et al., 2006; Pion et al., 2007b; Smed-Sorensen et al., 2005; Steinman et al., 2003;

Tschachler et al., 1987; Zambruno et al., 1991). In vitro studies have demonstrated that only a small fraction of DC populations areindeed infected and that maturation of DC dramatically impairs viral replication (Bakri et al., 2001; Cavrois et al., 2006b; Granelli-Piperno et al., 1998; Pion et al., 2007a; Smed-Sorensen et al.,2005). As explained before in this manuscript, HIV infection of DC depends on CD4 and the chemokine receptors CCR5 and CXCR4. DC however express relative small amounts of the viral receptors and co-receptors explaining partially why HIV only modestly infects DC when compared toCD4⁺ T cells (Rubbert et al., 1998; Turville et al., 2002). iDC mostly express CCR5 on their cell surface and very weakly CXCR4 (Granelli-Piperno et al., 1998). Recent investigations have however described that immature MDDC express similar levels of functional CXCR4 and CCR5 receptors. Fusion of X4-HIV with iDC is much less efficient compared to that of R5-HIV, making an Env-specific block early in the viral cycle the first step differentially hindering HIV infection of DC (Cavrois et al., 2006b; Pion et al., 2007a). It is of interest to note that these data perfectly fit clinical observations whereby individuals harboring a CCR5 homozygous mutation (CCR5- Δ32) remain protected from repetitive exposure to HIV, strengthening the potential selective

role DC could play during HIV sexual transmission as well as the major role for CCR5- mediated entry during HIV mucosal transmission (Huang et al., 1996; Liu et al., 1996;

Margolis and Shattock, 2006; Samson et al., 1996). Because of their antigen presenting

Characterization of HIV trafficking and DC-SIGN-mediated cytoskeleton rearrangement en the context of HIV transfer across dentritic cells-T lymphocytes infectious synapses

Thesis

Reference