Fate of MHC class II molecules in human dendritic cells

ARTICLE

MHC class II molecules expressed at the surface of antigen presenting cells mediate the presentation of peptides derived from exogenous antigens to CD4⁺ T lymphocytes. These molecules are assembled in the endoplasmic reticulum as nonamers composed of three alphaß dimers associated with one trimer of invariant chains. The invariant chains, chaperone molecules necessary for the correct folding of class II molecules in the endoplasmic reticulum, (i) prevent the alphaß dimers from associating with endogenous peptides in the endoplasmic reticulum and the Golgi complex and (ii) retain the class II molecules in the endoplasmic reticulum until nonamers are formed. The nonamers, guided by the invariant chains, then leave the endoplasmic reticulum and traverse the Golgi complex to reach the trans-Golgi network.

The formation of MHC class II-peptide complexes obviously requires the encounter of newly synthesized MHC class II molecules with (poly)peptides generated in the endocytic pathway. However, the route taken by class II molecules to reach the endocytic pathway remains a matter of controversy. According to some authors, newly synthesized class II molecules proceed directly to early [1-3] or late [4] endosomes, or to a prelysosomal compartment [5, 6]. In the endocytic pathway, proteases such as cathepsin S progressively cleave the luminal domain of the invariant chains [7]. Some fragments of the invariant chain nevertheless remain transiently associated with alphaß dimers, as is notably the case for a 10 kD fragment (p10) containing a region termed CLIP ("class II-associated invariant chain peptide"). Through interaction with the (poly)peptide-binding groove of alphaß dimers, CLIP not only mediates their association with the invariant chains but also prevents their loading of endogenous peptides. Once the CLIP region has been eliminated, a process catalyzed by HLA-DM molecules [8], (poly)peptides of exogenous origin can bind to the alphaß dimers. This association of (poly)peptides with alphaß dimers may take place, in the early endosomes for Guagliardi et al. [2], throughout the endocytic pathway for Castellino and Germain [3], or according to Peters et al. [5] in specific endocytic/lysosomal compartments denoted MIIC for "MHC class II compartments". Newly formed alphaß-peptide complexes then migrate to the cell surface where they become available for interaction with T cell receptors.

Dendritic cells and MHC class II molecules

Dendritic cells represent a family of antigen presenting cells [9] found in all tissues of the organism, in both non-lymphoid and lymphoid organs. In non-lymphoid organs, so-called "immature" dendritic cells play a sentinel role by capturing exogenous antigens, processing them and presenting the resultant immunogenic peptides associated with MHC class II molecules to mature T lymphocytes. Thus, these cells initiate a secondary immune response in non-lymphoid organs. However, under certain circumstances dendritic cells can migrate to the T cell areas of lymph nodes while they differentiate to develop a new phenotype and modify their functions. Surface expression of MHC class I and class II molecules increases, as does the expression of adhesion and costimulatory molecules playing an "accessory" role in antigen presentation. Meanwhile, their capacity to capture and process exogenous antigens is down-regulated. In the T cell areas of lymphoid organs, these "mature" dendritic cells may stimulate naive T lymphocytes and initiate a primary immune response against the antigens that had been "captured" in their tissue of origin.

What is known about the class II molecules expressed by dendritic cells? In human Langerhans cells, these molecules are found essentially at the cell surface and in lysosomal compartments of the endocytic pathway [10, 11]. The invariant chain molecules of dendritic cells display particularities affecting their distribution and biochemistry. Studies of the cellular distribution of the invariant chain have in fact revealed its unusually high expression on the surface of dendritic cells [12-14]. These cells produce the various invariant chain isoformsalpha, but in proportions which differ from those observed in other antigen presenting cells [15, 16]. Thus, mouse dendritic cells and in particular Langerhans cells produce four to five times more of the p41 isoform than B lymphocytes. This isoform has been shown to modulate antigen presentation by inhibiting proteolytic enzymes of the endosomal system [17]. In addition, the invariant chain molecules of murine dendritic cells are highly sialylated, which could influence their fate in the acidic compartments of the endocytic pathway through the negative charge conferred on MHC class II-invariant chain complexes [16].

How is one to explain the presence of the invariant chain at the surface of dendritic cells? In an attempt to answer this question, we studied the intracellular traffic of MHC class II and invariant chain molecules in human monocyte-derived dendritic cells. A unique route of transport for newly synthesized molecules was identified in these cells as compared to other antigen presenting cells, a pathway which may contribute to enhancing the efficiency of their antigen presentation and which will be discussed in the following paragraphs. However, the intracellular traffic of the invariant chain and MHC class II molecules displays further particularities depending on the "type" of dendritic cell.

Traffic of MHC class II molecules in human dendritic cells derived from monocytes

In man, the possibility of inducing the in vitro differentiation of peripheral blood monocytes into immature dendritic cells and of triggering their maturation as required [13, 18] has led to a better understanding of the fate of MHC class II molecules in immature dendritic cells and at the onset of maturation.

Traffic of MHC class II molecules in immature human dendritic cells

The differentiation of monocytes into dendritic cells is accompanied by important morphological, phenotypic and functional changes. Markers, of which the expression increases at the surface of in vitro differentiated cells, include not only HLA-DR molecules but also the invariant chain. In fact, the invariant chains present at the cell surface are not "free" but mainly associated with alpha and ß HLA-DR chains and, in immature dendritic cells, a large proportion of the newly synthesized alphaß-invariant chain complexes (>= 55 ± 13%) move directly and very rapidly in a continuous flux to the cell surface. On arrival at the surface, these complexes are spontaneously and rapidly internalized into coated pits and vesicles. The complexes then progress along the endocytic pathway, where the invariant chains are rapidly degraded while the first alphaß HLA-DR dimers are loaded with peptides, as demonstrated by their stability in the presence of SDS, before regaining the cell surface. Thus, 30 min after the start of their internalization, almost 75% of the alphaß-invariant chain complexes have been transformed into alphaß dimers, 20% of which are already to be found re-expressed at the cell surface. This movement towards the cell surface continues, with the result that 2 hrs following the start of their internalization, close to 75% of the class II molecules have regained the cell surface in the form of alphaß dimers [19].

What is the benefit to the dendritic cell of this passage by the cell surface of the alphaß-invariant chain complexes prior to their entry into the early endosomal system? Apparently, this sequence of events (Fig. 1) allows the dendritic cell to rapidly degrade the invariant chains soon after their arrival in the early endosomal compartments, where the alphaß HLA-DR dimers then load (poly)peptides [20]. In fact, the markedly slower transport of HLA-DR molecules from the early to the late endosomal compartments following depolymerization of the microtubules modifies neither the extent nor the kinetics of the conversion of alphaß-invariant chain complexes previously present at the cell surface into SDS-stable alphaß dimers [19]. Therefore, all along the endocytic pathway, in the early and probably in the later compartments, newly synthesized alphaß dimers can load antigenic (poly)peptides and migrate to the cell surface [19, 20]. The binding of polypeptides to MHC class II molecules is made possible by the structure of the peptide binding groove borne by the alphaß dimers. Since this groove, in contrast to that of MHC class I molecules, has no lateral limits, a single polypeptide can bind to several MHC class II molecules [20]. This association, starting in the early endosomal compartments and facilitated by the presence of HLA-DM molecules throughout the endocytic pathway [21], enables the preservation of antigenic determinants which would be destroyed by endosomal or lysosomal proteases if they were not protected by their position within the binding groove [20]. These enzymes can nevertheless digest the non-protected peptide fragments lying outside the peptide binding groove. Thus, through the particular traffic of their MHC class II molecules, dendritic cells derived from monocytes are able to optimize their capacity to present the diverse antigenic determinants of exogenous antigens.

On arrival at the surface of immature dendritic cells, by a process still poorly understood, alphaß dimers may recycle between early endosomal compartments and the cell surface [22], which allows them to load and present new peptides generated in the early endosomes. alphaß dimers do not however accumulate at the surface of immature dendritic cells and their half-life is no longer than about ten hours [22].

Fate of MHC class II molecules in mature human dendritic cells

Inflammatory stimuli such as TNF-alpha, LPS, CD40L and IL-1ß induce the maturation of dendritic cells derived from monocytes [18]. In immature cells in culture, these agents trigger: (i) a diminution of their capacity to capture antigens by macropinocytosis or through receptors which bind the mannose residues of proteins or immune complexes ; (ii) an increase in the surface expression of MHC class I and class II molecules and of molecules involved in cell adhesion (CD44, CD54 and CD58) and T cell costimulation (CD40, CD80 and CD86) ; (iii) appearance of the mature dendritic cell marker CD83 and (iv) at the functional level, a decrease in the capacity of these cells to process and hence to present exogenous antigens, together with an increase in their capacity to stimulate naive allogeneic T lymphocytes. In this way, from "immature" cells particularly efficient in the capture and treatment of antigens, dendritic cells evolve to "mature" cells specialized in the stimulation of naive T cells.

The increased expression of MHC class II molecules at the surface of maturing human dendritic cells has been shown to result from a number of factors: (i) a higher rate of synthesis of alphaß-invariant chain complexes at early time points [22] ; (ii) release of the class II molecules stored in the MHC class II-rich compartments [Haegel-Kronenberger et al., results submitted for publication] ; (iii) loss of the capacity to internalize cell surface alphaß dimers [22] and consequently (iv) prolongation of the half-life of class II molecules [22 and Haegel-Kronenberger et al., results submitted for publication]. An increase in invariant chain synthesis has been reported in dendritic cells treated with LPS or TNF-alpha, beginning as soon as one hour after stimulation and continuing for 10 to 16 hrs with a two to three fold higher rate of synthesis of class II molecules [22]. This could explain the rise in the expression of the invariant chain at the surface of dendritic cells during the first hours after triggering of cell maturation. However, results obtained in our laboratory suggest that increased neosynthesis is not the principal mechanism responsible for the surface induction of MHC class II molecules following TNF-alpha stimulation of dendritic cells.

The "disappearance" of the MHC class II-rich compartments 40 hrs after induction of dendritic cell maturation with TNF-alpha was reported for the first time by Sallusto et al. [18]. In order to better define the fate of the class II molecules which "disappear" in this manner, we studied the kinetics of HLA-DR expression at the cell surface and of the modifications affecting its intracellular distribution. The surface induction of HLA-DR alphaß dimers and their disappearance from internal compartments proved to be rapid and early phenomena. An increase in the level of expression of alphaß dimers at the cell surface was in fact already detectable only 30 min after TNF-alpha stimulation. This expression continued to increase regularly over the first six hours of maturation, during which time the lysosomal compartments seemed to maintain the presence of specific markers such as lamp-1, lamp-2, CD63 and CD68. What took place was a veritable "emptying" of the lysosomal compartments of their class II molecules. We then attempted to analyze the mechanism(s) of this emptying, firstly by testing the effects of drugs known to inhibit protein synthesis or different steps of intracellular traffic. The protein synthesis inhibitor cycloheximide did not affect the redistribution or surface induction of class II molecules at early timepoints following addition of TNF-alpha, but produced within 1 hr a sharp drop in the expression of the invariant chain at the cell surface. The alphaß dimers up-regulated on the surface of maturing cells at early stages of activation therefore represent pre-existing dimers originating from the intracellular MHC class II-rich compartments also containing lysosomal proteins. Brefeldin A, on the other hand, totally inhibited the surface induction of class II molecules and their disappearance from intracellular compartments, suggesting the involvement of a vesicular transport mechanism dependent on an exchange factor for ARF [23] in the emptying of MHC class II-rich compartments. Finally, depolymerization of the microtubules with nocodazole, but not of the actin filaments with cytochalasine D, had an inhibitory effect on this MHC class II redistribution. Does the emptying of MHC class II-rich compartments require their fusion with the cell membrane? To address this question, we followed the evolution of these compartments during early maturation by electron microscopy. In maturing cells, we observed (i) images suggesting the fusion of lysosomal compartments with one another and (ii) formation, at the expense of the lysosomal compartments, of tubular structures which appeared to establish connections between several such compartments. However, we did not observe images of fusion between lysosomal compartments and the cell membrane [D. Hanau, unpublished results]. These findings are consistent with other evidence that the export of MHC class II molecules from lysosomal compartments is a selective process. Thus, LPS or TNF-alpha stimulation does not lead to the active secretion of lysosomal enzymes by dendritic cells. Moreover, the lack of cell surface up-regulation of lysosomal membrane proteins such as lamp-2 during the early steps of maturation would argue against the direct fusion of MHC class II-rich compartments with the plasma membrane [Haegel-Kronenberger et al., results submitted for publication].

These results all point to the existence of a "specific" mode of transport of class II molecules from intracellular lysosomal compartments to the cell membrane, which would involve: (i) a vesicular transport mechanism dependent on an exchange factor for ARF ; (ii) microtubules of the cytoskeleton and (iii) the formation of interconnections within the lysosomal system of dendritic cells.

Prolongation of the half-life of HLA-DR molecules also contributes to increase the concentration of MHC class II molecules at the surface of mature dendritic cells. Thus, LPS treatment prolongs the half-life of HLA-DR at the surface of dendritic cells to over 100 hrs [22]. This has been attributed to a decrease after some hours and loss after 40 hrs of the capacity of maturing dendritic cells to internalize and degrade surface alphaß dimers [22].

In what measure is the neosynthesis of class II molecules modified in mature dendritic cells? The results of Cella et al. [22] suggest that neosynthesis ceases 24 hrs after the start of dendritic cell maturation in the presence of LPS. Our metabolic labeling experiments indicate on the contrary that neosynthesis persists when dendritic cell maturation is induced with TNF-alpha, even as long as 40 hrs after addition of the stimulating agent. Moreover, biochemical methods have enabled us to show that the intracellular traffic of newly synthesized class II molecules is not modified in this type of mature cell, the majority of alphaß-invariant chain complexes progressing first to the cell surface before internalizing to enter the endocytic pathway [H. Haegel-Kronenberger and
C. Saudrais, unpublished results]. In mouse bone-marrow-derived dendritic cells, control of invariant chain proteolysis by the cathepsin S inhibitor cystatin C has been shown to regulate the transport of MHC class II molecules during maturation [24]. Incomplete invariant chain proteolysis leads to the accumulation of class II molecules in the lysosomes of immature cells, while in mature dendritic cells the invariant chain is totally degraded and MHC class II molecules are efficiently transported to the cell surface. On the other hand, it has recently been shown that even in dendritic cells from mice bearing a genetically disrupted invariant chain, class II molecules can accumulate in lysosomal compartments. Moreover, after treatment with TNF-alpha these cells can export class II molecules from lysosomes to the cell surface [25], suggesting that the regulation of MHC class II transport here is not dependent on the invariant chain. This apparent discrepancy as to the influence of the invariant chain on the intracellular fate of MHC class II molecules might be related to the different MHC class II haplotypes of the mice used in the studies. However, neither study precludes the transport of pre-existing class II molecules stored in lysosomes to the cell surface upon activation. Internalization of the invariant chain is known to involve coated pits and vesicles [26]. Hence, it was of interest to look for the persistence of the process known as "receptor-mediated endocytosis" in mature dendritic cells. Using electron microscopy, it was possible to demonstrate that anti-CD1balphaalpha and anti-CD1calphaalpha antibodies continue, in mature as in immature dendritic cells, (i) to internalize through coated pits and vesicles and (ii) to progress to multilamellar compartments resembling in all morphological aspects, the MHC class II-rich compartments [D. Hanau, unpublished results]. The persistence of such multilamellar compartments in mature cells correlates with the findings of Calafat et al. [28], who have shown in transfected embryonic kidney cells that neosynthesis of class II alpha and ß chains is a necessary and sufficient condition to induce the appearance of multilamellar compartments of the MHC class II-rich type.

Thus, dendritic cells having undergone maturation in the presence of TNF-alpha appear to conserve: (i) the neosynthesis and unique transport pathway through the cell surface of class II molecules and (ii) multilamellar lysosomal compartments which no longer contain MHC class II molecules but nevertheless remain part of the endocytic pathway. However, this redistribution of MHC class II molecules, in mature dendritic cells derived from monocytes, from the lysosomal compartments to the cell surface, is in contrast to what has been observed in cultures of "blood dendritic cells".

Fate of MHC class II molecules in cultured human blood dendritic cells

Apart from monocytes capable, at least in vitro, of differentiating into dendritic cells, circulating blood contains a population of cells which are "already" dendritic. These cells are however present only in very low concentrations and represent no more than 0.1 to 1% of the total mononuclear cells of blood [12].

In freshly isolated blood dendritic cells, MHC class II molecules are found at the cell surface and in lysosomal "MHC class II-rich" compartments. As in the case of dendritic cells derived from monocytes, the expression of MHC class II molecules increases at the surface of these cells when they are maintained for 36 hrs in culture, but in contrast to cells differentiated from monocytes this expression also increases within the "MHC class II-rich" compartments. These compartments, which persist in culture and even become slightly more numerous, remain part of the endocytic pathway and maintain their content of lysosomal markers (CD63 and lamp-1) and HLA-DM molecules and their acidic nature [29]. Only the capacity to internalize a marker of endocytosis decreases in dendritic cells in culture, as does their capacity to present a heat shock protein antigen. Finally, one may note that both freshly isolated and cultured blood dendritic cells express the invariant chain at the cell surface and in the "MHC class II-rich" compartments. This persistence of the invariant chain at the surface of mature blood dendritic cells differs from observations in mature Langerhans cells.

Fate of MHC class II molecules in cultured Langerhans cells

Langerhans cells, the dendritic cells of the epidermis, spontaneously undergo maturation once dissociated from the surrounding keratinocytes, with resultant rapid modification of their phenotype and functions. Thus, the expression of MHC class II molecules at the surface of murine Langerhans cells in suspension increases during the first hours of culture to reach a plateau in 12 to 18 hrs [30]. However, while class II molecules persist on the surface of mature Langerhans cells, the invariant chain expressed on freshly isolated cells (partly at least as alphaß-invariant chain complexes [15]) disappears with maturation [14]. Interruption of the synthesis of alpha and ß MHC class II molecules and invariant chain 20 to 24 hrs after the onset of maturation is thought to be responsible for this disappearance [14-16].

A second factor would seem to contribute to the loss of the capacity of murine Langerhans cells to present exogenous antigens: the mature cells contain fewer early endosomes [31]. Similarly, after three days culture in the presence of GM-CSF, human Langerhans cells contain only small numbers of early endosomes and Birbeck granules. These two organelles have an acid pH in freshly isolated murine Langerhans cells, whereas this acidic character disappears after three days culture [31]. Loss of the capacity of certain organelles of the endocytic pathway to acidify has likewise been observed in mature human Langerhans cells by Girolomoni et al. [32], who showed that HLA-DR molecules, which in freshly isolated cells internalize and move to acid compartments, continue to internalize in mature cells but no longer reach acid compartments.

** Sugita et al. [27] have shown that CD1b molecules internalize in immature dendritic cells through coated pits and vesicles and we have recently observed the same to be true of CD1c molecules (unpublished results).

CONCLUSION

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