Langerhans cells are susceptible to measles virus infection and actively suppress T cell proliferation

ARTICLE

LC represent an immature type of DC located in various epithelial tissues including the skin and mucosae, and are considered as sentinels responsible for the uptake and processing of foreign antigens and activation of naive T cells upon migration to draining lymphoid organs. As such, they represent the first antigen-presenting cell type exposed to pathogens which penetrate via mucosal routes. Although LC are not very efficient at stimulating T cells due to the lack of expression of essential costimulatory molecules required for T cell activation, such as CD40, CD80 and CD86, they are highly phagocytic and are specialized in the capture and processing of pathogens and proteins into antigenic peptides. Stimuli, such as contact sensitizers or microbial infection, induce mobilization and maturation of LC, which migrate through afferent lymph to the T cell area of draining lymph nodes, where they constitute the mature, interdigitating DC expressing high levels of cell surface MHC class II, class I antigens and costimulatory molecules and are capable of activating naive T cell [1].

The efficiency of LC to initiate primary immune responses to pathogens or allergens, through activation of both CD4⁺ and CD8⁺ T cells has been thoroughly documented [2, 3]. On the other hand, there is as yet limited evidence that LC may be tolerogenic under certain microenvironment and/or pathophysiological situations.

Measles virus is a paramyxovirus which infects the host via the respiratory tract, and is subsequently disseminated to draining lymph nodes and then throughout the body, through mononuclear cells. Measles infection induces an immune response [4-6] which is efficient at clearing the virus, but which is associated with a dramatic immune suppression persisting months after the remission, affecting cell-mediated immunity to both recall and new antigens [7]. This immune suppression is responsible for the high mortality rate in endemic regions, due to increased susceptibility to opportunistic infections [8, 9].

Previous studies have suggested that antigen-presenting cells may be involved in MV-induced immune suppression. RNA encoding for measles virus fusion (F) protein has been detected in a limited number of circulating monocytes from patients with acute measles [10], and in vitro infection of monocytes with the measles virus has been seen to inhibit their ability to secrete IL-12 [11]. More recently, we and others have demonstrated that human DC differentiated in vitro from CD34⁺ cord blood progenitors with GM-CSF and TNF-alpha, or from peripheral blood monocytes cultured with GM-CSF and IL-4, could be productively infected with MV and lose their stimulatory property [12, 13]. However, there was no evidence that ex vivo, isolated LC, which are present in the airway epithelium at the portal of entry of MV, were susceptible to MV infection and pathogenesis.

This study provides evidence that the measles virus can infect LC irrespective of their maturation stage and turns functional LC into active inhibitory cells.

Materials and methods

Virus

MV vaccine strain Edmonston-B was grown and propagated in Vero cells (African green monkey kidney) and purified on a saccharose gradient. Virus stocks had a titer of 10⁷ PFU/ml. The wild type MV strain (LYS-1) isolated from PBMC of a patient with acute measles and passaged once on the permissive B95a marmoset cell line [14] had a titer of 3 x 10⁵ PFU/ml.

Epidermal cell suspension

Epidermal cells were isolated from normal skin of patients undergoing plastic surgery, as previously described [15]. Briefly, fragments of total skin were incubated for 1-2 hrs at 37° C in 0.05% of trypsin (Gibco, Lyon, France) in Hank's balanced medium supplemented with antibiotics. The epidermis was separated from the dermis, cut into 1 mm² fragments from which single cells were released by repeated pipetting. Epidermal cell suspensions were enriched for Langerhans cells (LC) by centrifugation for 20 min at 1,400 rpm over a Lymphoprep gradient. The cells recovered from the interface comprised around 30% of LC as revealed by morphology and expression of CD1a and HLA-DR (Fig. 1A) and will be referred to as LC. The cells recovered from the pellet were LC-depleted keratinocytes (Fig. 1B) and will be referred to as as KC. In some experiments LC were cultured for 24 hrs at 37° C in the presence of 10 ng/ml of rhGM-CSF to induce LC maturation and will be referred to as cultured LC.

Establishment of DC from CD34 progenitors

Dendritic cells were generated by culturing cord blood CD34⁺ progenitors in the presence of 100 ng/ml of rhGM-CSF (specific activity 2 x 10⁶ U/mg, Schering-Plough Research Institute, Kenilworth, NJ), 2.5 ng/ml (50 U/ml) of rhTNF-alpha (specific activity 2 x 10⁷ U/ml, Genzyme Corp., Boston, MA) and 25 ng/ml of stem cell factor in RPMI 1640 medium (Gibco, France) supplemented with 10% (vol/vol) FCS (Eurobio), 10 mM Hepes, 2 mM L-gutamine, 5 x 10^{­ 5} M 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin (referred to as complete medium), as previously described [16]. The cells, routinely collected at day 9, were composed of 60 to 90% of CD1a⁺ DC expressing MHC class-I, HLA-DR, DP, DQ, and CD46 molecules. These in vitro established CD1a⁺ DC will be referred to as cord blood-derived DC.

Infection of epidermal LC by MV

Freshly isolated LC, cultured LC and keratinocytes were infected by 1 hr incubation at 37° C with MV at a MOI of 0.1 PFU/cell, then washed and cultured at 37° C for 72 hrs, in Costar 6-well plates, at a density of 10⁶ cells/ml in 2 ml of complete medium supplemented with 100 ng/ml of rhGM-CSF.

May-Grunwald Giemsa staining

Analysis of LC syncytia was performed on day 3 of infection. The cells were cytocentrifuged onto glass slides and stained with May-Grunwald-Giemsa according to routine protocols.

Titration of measles virus produced by infected LC

LC suspensions were infected for 1 hr at 37° C with 0.1 PFU/cell of MV, washed 3 times and seeded at 2 x 10⁵ cells/well in triplicate wells of round-bottomed microplates in complete medium supplemented with 100 ng/ml of GM-CSF. Virus production in cell-free supernatants was assessed on days 0, 1, 2 and 3 of MV infection, by titration on Vero cells. The numbers of plaques were counted at various dilutions of supernatants and the results were expressed as the number of PFU/10⁶ cells.

Immunostaining of cell smears

Cells were cytocentrifuged for 5 min at 500 rpm onto glass slides and fixed in cold acetone for 10 min. Slides were washed in PBS supplemented with 5% human serum and incubated for 30 min with either a polyclonal rabbit anti-mouse antibody specific for S100 (dilution 1/400) (Dako, Trappes, France) (or normal rabbit serum as control) or with the NP specific mAb 25Cl [17] (or normal mouse serum as control). Specific staining was revealed using a biotinylated Fab'2 fragment of goat anti-rabbit IgG antibody (dilution 1/400) (Vector, Biosys, Compiègne, France) or a biotinylated Fab'2 fragment of goat anti-mouse IgG (H + L) (dilution 1/50) (Caltag, Tebu, France), respectively. The slides were then incubated with streptavidin-peroxidase (ABC kit, Dako, France). The enzyme activity was developed by AEC substrate and H₂O₂. The slides were washed and counterstained with hematoxylin.

FACS analysis

Indirect immunofluorescence analysis was performed according to standard techniques. Briefly, cells were first incubated for 20 min on ice with 5% normal human AB⁺ serum to saturate Fc receptors, then stained with the following specific antibodies: the anti-MV-HA mAb55 (mouse IgG2b) [18], an anti-CD1a-FITC mAb (Ortho Diagno System), an anti-DR-FITC Mab (Dako), anti-CD80-PE (Becton Dickinson), anti-CD86-PE (Pharmingen), anti-CD83 (Immunotech, Marseille, France) [19]. Specific staining was revealed by an FITC-conjugated goat F(ab')2 anti-mouse IgG (H + L) antibody (Zymed, Tebu, France). Controls included normal mouse IgG and FITC/PE- conjugated mouse IgG. Flow cytometry analyses were performed using a FACSstar plus cytofluorimeter (Becton Dickinson) equipped with Lysis II software.

Purification of naïve CD4⁺ T cells from human peripheral blood

Mononuclear cells were isolated from adult peripheral blood by Ficoll Paque gradient centrifugation [20]. Naive CD4⁺ T cells were purified by immunomagnetic depletion of mononuclear cells using a cocktail of mAbs including IOM2 (CD14), ION16 (CD16), B8 12.2 (HLA-DR) (from Immunotech, France), OKT8 (CD8) (Ortho), UCHL1 (CD45RO) (Dako), NKH1 (CD56) (Coulter), 4G7 (CD19) (Becton Dickinson) and mAb 89 (CD40) [21] (a gift from C. Caux, Shering-Plough, Dardilly). After two rounds of bead depletion, the purity of CD45RA⁺ CD4⁺ T cells was routinely higher than 95%. The T cells were frozen at 5.10⁶ cells per vial in PBS, 90% FCS and 10% DMSO until use.

Allogeneic mixed lymphocyte reaction

Various numbers of LC, either mock-infected or MV-infected were used as stimulatory cells in allogeneic MLR with naive CD45RA⁺ CD4⁺ T cells (2.10⁴ cells/well) in triplicate round-bottomed microtiter plates (Falcon) in complete RPMI medium.

In some experiments, MV-infected and mock-infected LC were first UV-irradiated (0.25 J/cm²) to inactivate the virus within the cells and then added to co-cultures containing either 10⁴ uninfected LC or DC derived from cord blood progenitors, and 2.10⁴ naive allogeneic CD4⁺ CD45RA⁺ T cells. Cultures lasted for 6 days and were pulsed with 1 µCi of ³H-thymidine for the last 16 hrs of culture, harvested and thymidine incorporation was counted using a ß-counter (Wallac). The results are expressed as cpm ± SD of quadruplicate wells.

Results

Phenotype of fresh and cultured epidermal cells

Freshly isolated LC suspensions contained 20-30% of CD1a⁺ HLA-DR⁺ LC (Fig. 1A), which were virtually absent from keratinocyte suspensions (Fig. 1B). Phenotypic studies confirmed that fresh LC had a phenotype of immature DC, characterized by high levels of CD1a, but low to undetectable levels of CD83 and of the costimulatory molecules CD80 and CD86 (Fig. 2A). Alternatively, LC cultured for 24/48 hrs in the presence of GM-CSF had a mature DC phenotype manifested by decreased CD1a density and enhanced expression of HLA-DR, CD80, CD86 and CD83 molecules (Fig. 2B). Keratinocyte-suspensions cultured for 24 hrs with GM-CSF were still devoid of DR⁺ and CD1a⁺ cells, thus confirming that they were not contaminated with LC (not shown).

Measles virus infects LC and induces syncytia formation

Infection of either fresh or cultured LC suspensions with MV, resulted in cell fusion and syncytia formation, observed within 3 and 5 days of infection with the vaccine (Edmonston, Hallé) (Figs. 3a and 3b) and the wild type LYS-1 (not shown) MV strains, respectively. Giant multinucleated cells comprising 20 or more nuclei (Figs. 3e and 3f), developed with similar kinetics after infection of fresh or cultured LC (not shown). In contrast, keratinocytes suspensions never gave rise to syncytia up to day 5 of cultured with MV (Figs. 3c and 3d).

Since LC-enriched suspensions were contaminated with keratinocytes, immunocytochemical stainings for cytoplasmic NP and S100 antigen were carried out to demonstrate that syncytia formation resulted from LC fusion. LC suspensions were either uninfected (Figs. 4a and 4d) or infected with MV (Figs. 4b, c, e and f) and analyzed on day 3 of infection. In uninfected cultures, LC expressing the S100 protein could be identified as single cells and as clusters with S100 negative keratinocytes (Fig. 4a), and as expected, did not stain for cytoplasmic NP (Fig. 4d). In MV-infected cultures, S100⁺ LC were present as single cells and as syncytia (Figs. 4b and c), both expressing cytoplasmic NP (Figs. 4e and f). Keratinocytes contaminating the LC suspensions, identified by their typical stone-like morphology, their central nucleus and lack of S100 expression (Fig. 4b), lacked NP (Fig. 4e) although they were found associated with S100⁺ syncytia (Fig. 4c).

FACS analysis confirmed that LC, but not keratinocytes, were infected with MV. As shown in Figure 5, MV-infected LC suspensions contained 34% of DR⁺ and HA⁺ cells present as single cells, while no HA⁺ cells could be found in keratinocyte suspensions.

These data show that MV-infected LC coexpressing S100 and NP are necessary for syncytia formation and that keratinocytes, although not directly infectable by MV, may also be present in LC syncytia.

LC infection by MV is productive

We next examined whether infection of LC with MV yielded infectious virus production. The supernatant of freshly infected LC-enriched suspensions was titrated at various times after infection for the presence of infectious MV with a plaque assay and the permissive Vero cells. LC suspensions could efficiently replicate MV and yield infectious particles with a titer of 3.10⁴ PFU/10⁶ cells which peaked on day 3 of infection. Parallel cultures of Vero cells infected with MV showed a maximal virus titer of 10⁵ PFU/10⁶ cells (Fig. 6).

MV-infection abrogates the ability of LC to stimulate the proliferation of naive T cells in allogeneic MLR

We next asked whether MV infection would affect the ability of LC to activate naïve allogeneic CD4⁺ T cells. Cultured-LC, either mock-infected or MV-infected, were harvested on day 3 of infection and added in various numbers to naive allogeneic CD45RA⁺ CD4⁺ T cells. T cell proliferation was analyzed 6 days later. As shown in Figure 7A, as few as 100 mock-infected cells (containing roughly 30% LC, i.e. 30 LC) were efficient at stimulating T cell proliferation. In contrast, up to 10⁴ MV-infected cells (i.e. comprising around 3.10³ LC) were unable to induce T cell proliferation. These data showed that MV-infected LC could no longer support the proliferation of allogeneic CD4⁺ T cells.

MV-infected LC actively suppress proliferation of naive allogeneic T cells independently of infectious virus release

Because infectious MV produced by LC could directly infect T cells and block their proliferative capacity, we next examined whether inhibition of T cell activation could be induced by day 3 MV-infected LC, in which virus replication was blocked by UV-treament. Since UV treatment abrogated the ability of uninfected control LC to stimulate allogeneic MLR (data not shown), we set up a coculture system in which day 3 UV-treated MV-infected or mock-infected LC were added in graded numbers to uninfected LC and allogeneic T cells. UV-treatment of MV-infected LC inactivated virus replication inside the cells and secretion of infectious virions, as confirmed by the plaque assay performed with either cell-free supernatant or whole cultures (virus titer of 0 PFU/ml on day 0 (1 hr), 1 or 2 after infection). Addition of uninfected UV-irradiated LC did not affect the proliferation of naive allogeneic CD4⁺ T cells in response to uninfected LC. In contrast, UV-treated MV-infected LC completely blocked T cell proliferation in allogeneic MLR between uninfected LC and T cells (Fig. 7B). These data show that MV turns LC into efficient tolerogenic cells, which can block T cell proliferation in response to allogeneic stimuli by a mechanism which is independent of virus production.

Suppression mediated by MV-infected LC is independent of the maturation stage of LC at the time of infection and is antigen non-specific

We next examined whether the induction of inhibitory LC by MV infection was dependent upon the maturation stage of LC at the time of infection. We thus compared the inhibitory effect of UV-treated LC that had been MV-infected 3 days before, as either fresh or cultured LC (Fig. 2). Addition of as few as 100 UV-treated MV- infected fresh (Fig. 8A) or cultured (Fig. 8B) LC to MLR consisting of uninfected DC (derived from cord blood CD34⁺ progenitors) and allogeneic T cells, completely abrogated the proliferative response of the T cells. Addition of up to 10⁴ UV-treated mock-infected fresh or cultured LC did not affect the magnitude of T cell proliferation in response to DC, indicating that active inhibition by MV-infected DC was not merely due to a steric hindrance phenomenon. Taken together these data indicated that LC become actively suppressive after MV-infection, irrespective of their maturation stage at the time of infection. The observation that MV-infected LC could tolerize T cells responsive to DC originating from an HLA-disparate third party donor further indicated that the mechanism of inhibition is antigen non-specific and MHC-unrestricted.

Discussion

In this study, we show that LC, isolated from normal human epidermis can be productively infected in vitro with wild type and vaccine strains of MV, as shown by the presence of both S100⁺ LC as single cells expressing cell surface HA and cytoplasmic NP, and as S100⁺ NP⁺ LC syncytia. That LC, but not keratinocytes contaminating LC suspensions, were infected with MV was further confirmed by the observation that pure suspensions of DR^­ CD1a^­ keratinocytes cultured for up to five days following MV-infection lacked expression of MV-NP and MV-HA and never gave rise to syncytia. The observation that immature LC and mature LC are equally susceptible MV targets extends previous findings obtained with in vitro-derived DC (12, 13) to LC isolated ex-vivo from epithelial tissues.

The epithelial lining of the conductive airways is populated by a network of LC [22], which migrate to T cell areas of lymphoid tissues draining the upper respiratory tract. Clinical and pathological findings, as well as experimental studies in monkeys, have indicated that infection is initiated by invasion of MV through the respiratory epithelium and primary multiplication of the virus in the pharynx and tonsils [23, 24]. Interestingly, syncytia of reticuloendothelial cells (i.e. Warthin Finkeldley cells) have been identified during the prodrome, in the subepithelium of these tissues where LC are located [25]. These giant cells are considered as a major virus reservoir responsible for systemic virus spreading through the blood to various mucosal epithelia (i.e. the trachea, the lung, the conjunctiva, the mouth and to the skin), which occurs during the rash. Although the cell type which actively transports the virus from the epithelium of the upper respiratory tract to the draining lymphoid tissues has not been identified, our data support the hypothesis that LC may be infected by MV invading the epithelium and contribute to both the syncytia identified in the airway epithelium and to virus transport to the lymph nodes. MV-infected LC which remain as single cells could also transmit infection to mature interdigitating DC and/or lymphoid cells, upon migration to the lymph nodes, further amplifying systemic spread of the virus.

The functional consequences of MV infection of either fresh or cultured LC are threefold. First, MV replication in LC abrogated their stimulatory function towards naive allogeneic CD45RA⁺ T cells. This is most likely due to the release of infectious virus, previously reported to block T cell proliferation by an arrest in the G1 phase of the cell cycle [26]. Secondly, and more interestingly, MV infection induces the switch of LC from an immunogenic to an active inhibitory cell. Indeed, we showed that, when replication and secretion of infectious virus by infected-LC were blocked by UV-treatment, infected-LC exerted a potent inhibition of allogeneic T cell proliferation in response to uninfected LC in as much as as few as 10² UV-treated MV-infected LC completely prevented the allostimulatory effect of 10⁴ uninfected DC. The mechanism involved in this active suppression most likely involves cell-cell contact because UV-irradiated supernatant (free of infectious virus) from MV-infected DC is unable to induce inhibition of allogeneic MLR (data not shown). That LC become tolerogenic irrespective of their maturation stage at the time of infection may be related to the ability of MV to induce DC maturation as revealed by upregulation of HLA-DR, CD83 and the costimulatory molecules CD80 and CD86 [27; and D. Kaiserlian unpublished data].

It is not clear at present whether MV-infected LC can transduce negative signals through a direct interaction with the T cells or an indirect interaction with uninfected stimulatory LC. It is unlikely that inhibition of T cell responsiveness to uninfected LC is due to the anergy of alloreactive T cells through cognate MHC/TcR interaction, because tolerance can be induced even when MV-infected LC are MHC disparate from the uninfected-DC used as stimulatory cells in the MLR. Nonetheless, it remains possible that infected-LC anergize the T cells through non-cognate interaction. In this respect, previous studies have suggested that HA and F expressed at the surface of MV-infected PBMC or transfected cells are necessary and sufficient to transduce negative signal blocking T cell proliferation [26, 28] possibly through binding to the cellular MV receptor, CD46 [29]. However, this does not seem to be the case in our system since UV-treated Vero cells infected with a vaccinia recombinant encoding MV-HA and F, coexpress HA and F at the cell surface, yet are inefficient at blocking DC/T MLR (data not shown). This suggests that, although HA and F may act as adhesion molecules allowing interaction of a limited number of infected LC with T cells (or uninfected DC), other molecules, possibly upregulated by MV infection, may be responsible for transducing the inhibitory signal.

The present study provides the first demonstration that ex vivo isolated LC can replicate MV and become actively suppressive, suggesting that DC infection may play a central role in virus spread and immune suppression of cell-mediated immune responses during natural infection with measles. LC have been implicated as the cells carrying HIV from the skin or mucosa to the lymph nodes during virus transmission in primates [30]. DC syncytia in tonsils of seropositive, asymptomatic HIV patients have also been reported [31]. We propose that during natural MV infection, epithelial DC of the airway epithelium, which are similar to epidermal LC, may represent the initial target of the measles virus responsible for virus transport to draining lymph nodes and induction of antigen non-specific immune suppression through active tolerogenic signals, independent of virus secretion. Further studies are required to investigate the precise mechanism of the suppressive effect of LC in our system and to determine the relative contribution of HA and F and of inducible DC molecules in the active suppression mediated by infected DC.

CONCLUSION

Acknowledgements

We are grateful to doctors and colleagues from clinics and hospitals in Lyon who provided us with umbilical cord blood samples and human skin from plastic surgery.

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