Coalescence of B-cell receptor and invariant chain-MHC II in a raft-like membrane domain

Aus dem Zentrum Anatomie

Institut für Zellbiologie im Zentrum Anatomie (Direktor: Prof. Dr. rer. nat. E. Ungewickell)

der

Medizinischen Hochschule Hannover

Coalescence of

B-cell receptor and invariant chain-MHC II in a raft-like membrane domain

Dissertation

zur Erlangung des Doktorgrades der Medizin

in der Medizinischen Hochschule Hannover

Vorgelegt von Julian Till Hauser

aus Hannover

Hannover 2014

Angenommen vom Senat der Medizinischen Hochschule Hannover am 09.06.2015.

Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover

Präsident: Prof. Dr. med. Christopher Baum

Betreuer der Arbeit: Prof. Dr. rer. nat. Ernst Ungewickell Dr. rer. nat. Robert Lindner

Co-Betreuer der Arbeit: Prof. Dr. rer. nat. Reinhard Schwinzer

Referent: Prof. Dr. med. Johannes Gessner

Korreferent: Prof. Dr. med. Dr. rer. nat. Andreas Schmiedl

Tag der mündlichen Prüfung: 09.06.2015

Promotionsausschussmitglieder: Prof. Dr. rer. nat. Evgeni Ponimaskin

Prof. Dr. rer. nat. Rita Gerardy-Schahn

Prof. Dr. rer. nat. Reinhard Schwinzer

Diese Dissertationsschrift basiert gemäß § 3 Absatz 3 der Promotionsordnung der Medizinischen Hochschule Hannover auf folgender Publikation:

Hauser JT, Lindner R. Coalescence of B cell receptor and invariant chain MHC II in a raft-like membrane domain. J Leukoc Biol. 2014 Nov;96(5):843-55.

Die Dissertation wurde im Rahmen der strukturierten Doktorandenausbildung

angefertigt.

TABLE OF CONTENTS

1. Introduction

1.1 The B-cell receptor (BCR) 1-3

1.1.1 Signaling 3-4

1.1.2 Internalization 4-5

1.2 Major histocompatibility complex class II (MHC II)

and invariant chain (Ii) 5-6

1.3 Relationship between BCR-mediated antigen delivery and

Ii-mediated MHC II delivery pathways 6-7

1.4 Membrane domains and lipid rafts 7-8

2. Working hypothesis

3. Synopsis of results

4. Publication

5. Discussion

5.1 Weak association of clustered BCR with

detergent resistant membranes (DRMs) 27-28

5.2 Clustering of invariant chain:

not only an artificial approach 28-31

5.3 A novel role for the pro-inflammatory cytokine MIF

(Macrophage migration inhibitory factor) 31-32

6. Summary

7. References

8. Curriculum vitae

9. Erklärung nach § 2 Abs. 2 Nr. 6 und 7 Promotionsordnung

10. Danksagungen

1. Introduction

1

1. Introduction

B-cells are well known as professional antigen presenting cells (APCs). They differ from other professional APCs, like macrophages or dendritic cells, by the expression of a specialized antigen receptor, the B-cell receptor (BCR), on the cell surface. This receptor serves to bind antigens (Ag)s, which are, by definition, molecules recognized by the immune system. The BCR is extremely variable within the population of B-cells. Every B-cell usually expresses BCRs of only one type of specificity. The BCR serves to deliver bound Ag to intracellular compartments that are capable of processing the antigen and loading the resulting peptide fragments onto nascent major histocompatibility complex class II (MHC II) molecules. These molecules are specialized presentation platforms that are transported to the cell surface once they are stably associated with suitable peptides. There, MHC II-bound peptides can be recognized by CD4⁺ T-helper cells expressing a specific T-cell receptor. During this process the B-cell becomes activated, which results in the commencement of affinity maturation in germinal centers - a process that leads to the production of long-lived memory B-cells and plasma cells. The latter secrete the high-affinity Ag-binding module of the BCR in an altered, soluble form, which is called antibody (Ab) or immunoglobulin (Ig) (1).

1.1 The B-cell receptor (BCR)

B-cells play a key role in the adaptive immune system by producing a type of antigen recognition molecule with an extremely wide variety of antigen specificity. In its soluble form this specialized glycoprotein consists of two large heavy and two small light chains forming a Y-shaped molecule. The tips of the heavy and light chains form an antigen-binding groove, the paratope, which is unique and specific to a cognate structure on the antigen, the epitope. The antigen recognition event might be envisioned as a key-lock-mechanism. The specificity of the "lock" (the paratope) is encoded by the vast variety of different tips of light and heavy chains generated by recombination of gene segments (see below) and also by the diversity brought about by the random combination of light and heavy Ig chains in an 1:1 complex. Antibodies show only limited variability outside of their antigen binding regions:

there are five isotypes known as IgA, IgD, IgE, IgG, and IgM with further subtypes for the IgA and the IgG classes (2). Ig molecules can be produced as soluble proteins by plasma cells, but they also form the main component of the antigen-specific receptor of B-cells, the B-cell

1. Introduction

receptor (BCR). In B-cells they are expressed in an alternative form with a transmembrane anchor and a short cytoplasmic tail sequence. The BCR facilitates processing of cognate antigens and the presentation of antigen-derived peptides to CD4+ T-cells at concentrations far below those required for B-cells displaying a BCR with no affinity for cognate antigen (3).

The generation of highly diverse B-cell receptors is tightly coupled to B-cell development. It is initiated in the bone marrow by the differentiation of common lymphoid progenitor cells to pro-B-cells. In these cells, the recombination of three types of gene segments on one of the two Ig heavy chain allelic loci (V_H, D_H and J_H segments) is triggered. First, an arbitrary D_H gene segment is recombined with an arbitrary J_H segment followed by the recombination of one of about 60 V_H segments with the already rearranged D_HJ_H-segments. At this time, the cell enters the pre-B-cell stage, which is characterized by the expression of a low amount of pre-B-cell receptor on the cell surface. This receptor consists of an Ig heavy chain (µ-subtype) transcribed from the newly rearranged heavy chain locus in a complex with an invariable

"surrogate" light chain and signal transduction components (see below). The surface expression of a functional pre-B-cell receptor signalizes successful VDJ recombination and allows the pre-B-cell to enter the next developmental stage, in which Ig light chain genes are rearranged. Since the light chain gene locus lacks D segments, rearrangements occur by V_L to J_L joining. Similar to the heavy chain recombination process, only one of the light chain allelic loci is rearranged at a time, thus preventing the generation of two different light chain specificities. In addition, during the process isotypic exclusion takes place, i.e. the selection of only one type of light chain - κ or λ. Assembled as a fully functional immunoglobulin consisting of two heavy and two light chains with a unique antigen specificity, the complex is expressed on the cell surface as the B-cell receptor (IgM subtype), now marking the immature B-cell stage. Appropriate signals by the pre-BCR are crucial for proliferative expansion and survival, i.e. positive selection. By contrast, during the negative selection process, immature B-cells are tested for auto-reactivity. Binding of the BCR to self-antigens leads to clonal deletion or receptor editing. To ensure self-tolerance, only B-cells that are non-reactive to self-antigens are allowed to mature (4). Having passed through positive and negative selection processes and leaving the bone marrow, the now mature B-cell expresses membrane-anchored IgM and IgD molecules on the cell surface that share the same antigen specificity. Together with Ig heavy chain class switch recombination that determines which

1. Introduction

3 of the five Ig isotypes (IgA, IgD, IgG, IgM and IgE) is produced, every B-cell is capable of either expressing its unique Ig on the plasma membrane as a part of the BCR-complex or, after BCR-mediated activation and T-cell-assisted differentiation to plasma cells, as secreted soluble antibody (1, 5).

The immediate functions of membrane-anchored immunoglobulin, as to be discussed in the chapters below, are signaling and internalization of bound antigen. To exert these functions, membrane-anchored immunoglobulin needs to interact with cytosolic molecules like kinases or adaptor proteins. In contrast to IgG that behaves differently due to an internalization motif in its cytosolic tail, membrane-anchored IgM and IgD are not directly linked to the cell’s signaling and internalization apparatus (6, 7), because they are invested with only very short cytosolic segments (three amino acid residues). For this reason, these BCRs are dependent on the noncovalent association with the disulfide-linked Igα-Igβ heterodimer, which functions as a signaling and internalization module (8, 9). For both of these functions so-called immunoreceptor tyrosine-based activation motifs (ITAMs) that reside in the cytoplasmatic tails of Igα- and Igβ (10-12) are critical. Depending on whether these sequences become tyrosine-phosphorylated upon antigen binding or not, they serve as signaling initiators or internalization motifs, respectively (11).

1.1.1 Signaling

It has been shown that B-cell responses differ depending on the valency of the activating antigen: monovalent binding of the B-cell receptor induced receptor activation but failed to promote antigen presentation, whereas polyvalent ligation of the BCR by (oligomerized) antigen promoted both, receptor activation and antigen presentation (13). Evidence was provided that the inefficient presentation of monovalent antigen could be overcome by membrane anchorage of the antigen in trans (i.e. on another cell) (14). This is in agreement with observations that the display of unprocessed membrane-associated antigen by other APCs like macrophages and follicular dendritic cells in vivo appear to play a critical role in initiating B-cell response (15, 16). Membrane-bound antigen has been shown to trigger BCR clustering and the formation of an immunological synapse, a transient contact between two immune cells consisting of concentrically ordered yet dynamic arrays of membrane proteins (17, 18). Within such a synaptic structure, microclusters of BCR, antigen, and signal

1. Introduction

transduction molecules are formed (19, 20). Evidence has been provided that a special membrane environment termed "lipid rafts" is transiently assembled in these regions (21).

The signaling cascade is initiated upon the BCR’s translocation into this specialized membrane environment. It is thought that this step is crucial for the recruitment of Scr- Family kinases members, such as Lyn in particular, which is enriched in lipid rafts and associates more stably with the BCR upon antigen binding (21). By doing so, Lyn is capable of phosphorylating the ITAMs located in the cytoplasmatic tails of the Igα-Igβ heterodimer. This event plays a key role in the recruitment of Syk tyrosine kinase to the phoshorylated ITAMs and its activation. Active Syk and Lyn initiate the formation of a complex structure called the

"signalosome", which consists of scaffolding proteins, GTP-binding proteins, lipases and kinases that link BCR ligation to several intracellular signaling pathways (MAPK, PI-3 kinase, Ca²⁺/PKC and NFκB pathways). In combination with other effector mechanisms and additional supportive T helper cell-signals, activation, differentiation and clonal expansion of antigen-specific B-cells are triggered (22, 23).

Qualitatively different signaling cascades have been demonstrated for BCR composed of IgG in contrast to those BCRs composed of IgM or IgD. It was found that, in IgG positive B-cells CD22-mediated signal inhibition was diminished by preventing CD22 phorphorylation. This effect was dependent on the cytoplasmatic tail of IgG. (24). In addition, a tyrosine residue in the conserved, 28 amino acids long IgG tail serves to recruit growth-factor-receptor-bound protein 2 (GRB2) resulting in enhanced B-cell proliferation through sustained kinase activation and generation of second messengers (25). This may explain why IgG class- switched B-cells have the edge over unswitched B-cells (26, 27).

1.1.2 Internalization

Although it is known that signaling is highly dependent on the degree of BCR cross-linking (28) different levels of BCR engagement do not appear to alter the rate of Ag-BCR endocytosis (29). Clustering of the BCR-antigen complexes and translocation into lipid rafts is accompanied by clathrin-dependent or -independent endocytosis. In a study BCR uptake was reduced by approximately 70% in B-cells that were conditionally deficient in clathrin heavy chain expression (30). Actin or raft antagonists were found to block the residual, clathrin- independent BCR internalization. More evidence for an involvement of actin in BCR

1. Introduction

5 internalization was provided by Song and co-workers, who studied the effect of the actin recruitment protein N-WASp on BCR internalization (31). However, published data from other laboratories demonstrate that BCR internalization of cognate antigen is dependent on dynamin (32) and occurs mostly via a lipid raft-independent mechanism (33). In agreement with this notion, a non-phosphorylated ITAM motif of Igβ has been shown to function as the major binding site for the AP2 adaptor complex (34), which serves to recruit clathrin to receptors in the plasma membrane (35). Furthermore, the E3-type ubiquitin ligase Itch has been demonstrated to be essential for BCR internalization via clathrin-coated pits, although the ubiquitinylation of IgM heavy chain and Igβ appear to be dispensable for this process.

Ubiquitinylation of conserved lysine residues on these chains appears to be mandatory for proper trafficking of the BCR within endocytic compartments (36).

1.2 Major histocompatibility complex class II (MHC II) and invariant chain (Ii)

The main function of MHC II is to present exogenous antigen, internalized by antigen presenting cells, to class II-restricted CD4⁺ T-cells (reviewed in (37)). The heterodimeric MHC II molecule consists of an α- and β-chain and is synthesized in the rough endoplasmatic reticulum (rER), where it associates with invariant chain (Ii). Ii is assembled as a trimeric chaperone that assists newly synthesized MHC II in folding and prevents endogenous peptides from binding to the MHC II in the rER (38). Ii is thought to form a nonameric complex with MHC II molecules, comprising the Ii-trimer and three MHC II α- and β-chains (39), although this structural arrangement has been questioned recently (40). Due to an endosomal targeting and retention motif in its cytoplasmatic tail, Ii is also responsible for sorting of the associated MHC II molecules from the trans-Golgi network to endosomal compartments (38, 41, 42). Only a small fraction of invariant chain-MHC II (Ii-MHC II) complexes is present at the cell surface of B-lymphocytes, which appears to be derived from early endosomes via a recycling pathway (41). In the acidic environment of late endosomes / lysosome-like compartments, Ii is degraded. The lysosomal cysteine protease Cathepsin S performs the last cleavage leaving a small peptide fragment called class II invariant chain peptide (CLIP) that still seals the antigen binding groove of MHC II. Catalyzed by a lysosomal MHC II-like molecule, H2-M (the murine equivalent of HLA-DM, a chaperone and MHC II peptide loading editor), CLIP is exchanged for antigenic peptides generated in the acidic environment of the lysosome (reviewed in (43)). The degradation of Ii also leads to a

1. Introduction

removal of the endosomal targeting motif of Ii from MHC II allowing the latter to be transported to the plasma membrane upon peptide loading.

1.3 Relationship between BCR-mediated antigen delivery and Ii-mediated MHC II delivery pathways

After internalization, the BCR-antigen clusters are targeted via early endosomes to Ii-MHC II- enriched multivesicular bodies (MVBs), which newly form after BCR engagement (44, 45).

MVBs are mostly found in a centralized location containing all essential components for antigen processing, namely newly synthesized, invariant chain-bound MHC II, H2-M and proteases in an acidic environment, with members of the cathepsin family being most prevalent. Here, the BCR is degraded releasing the antigen, which is also proteolytically processed into short fragments to be loaded onto newly synthesized MHC II (46). In non- activated B-cells, MHC II molecules mostly accumulate at the cell surface and in an intracellular pool of tubulo-vesicular structures that are distinct from lysosomal compartments defined by the presence of lamp-1 and H2-M. BCR stimulation induces the transient intracellular accumulation of MHC class II in MVBs to which H2-M is recruited (44, 47) Furthermore, BCR engagement also triggers the temporary downregulation of cathepsin S activity and this supports the transient accumulation of Ii-MHC II complexes in MVBs (48).

In addition, it was reported that the non-processive motor protein myosin II is associated with Ii-MHC II complexes. Upon polyvalent BCR engagement myosin II became phosphorylated on its light chains and this activation triggered the convergence of myosin- bound Ii-MHC II-complexes with antigen-bound BCR in late endocytic MVB compartments.

(49).

In conclusion, there is substantial experimental evidence that the BCR delivers its cognate antigen to the MHC II peptide loading pathway (46, 49, 50) as summarized in the following scheme:

1. Introduction

7 Fig. 1: Cognate soluble polyvalent antigen or membrane-anchored antigen binds and clusters the BCR-Igα-Igβ-complex.

This is followed by translocation into a specialized membrane environment (lipid rafts), initiation of a complex downstream signaling cascade and, finally, endocytosis. Via early endosomes the BCR-antigen complex reaches late endosomes/multivesicular bodies (MVBs). The antigen is degraded into short fragments by proteases within an acidic environment. MHC II and invariant chain are synthesized in the rough ER and after assembly to nonameric structures (Ii-trimer + 3 MHC II molecules (α- and β-chain)) they travel through the Golgi and Trans-Golgi-network (TGN) to the endosome-pathway. Finally, in late endosomes/multivesicular bodies, Ii is cleaved into the p11-fragment followed by degradation by cathepsin S leaving the CLIP-fragment in the MHC II-antigen-binding groove. H2-M catalyzes the exchange of CLIP for antigenic peptides derived from BCR-bound material. The MHC II-peptide complex is exported to the cell surface for antigen presentation and recruitment of T-cell help.

Note: Ii-MHC II-complexes are present at the cell surface due to a recycling pathway through early endosomes and are associated with lipid rafts (41, 51).

1.4 Membrane domains and lipid rafts

There are two principal groups of membrane domains: protein-based membrane domains and lipid-based membrane domains.

Protein based membrane domains are organized by either extracellular, cytoplasmic or membrane protein scaffolds. Galectins are the most prominent members of extracellular protein scaffolds. They form membrane domains by cross-bridging the extracellular domains

1. Introduction

of membrane glycoproteins. The best known intracellular protein scaffolds are the clathrin- coated structures, which are critical for receptor-based internalization of various ligands into eukaryotic cells amongst other functions. Membrane proteins such as tetraspanins, caveolin and members of the SPFH (Stomatin/Prohibitin/Flotillin/HFLK) protein family have been described to serve as intra-membrane scaffolds (reviewed in (52)).

Lipid based membrane domains (syn.: lipid rafts, membrane microdomains) are characterized as liquid-ordered membrane nanoclusters that are enriched in cholesterol and sphingolipids. They often contain and are additionally characterized by a subset of GPI- anchored or double acylated proteins (53, 54). In living cells these nanoclusters are highly heterogeneous, short-lived (10-20ms) and smaller than 20nm in diameter (55-57). One of their main functions is thought to be the compartmentalization of cellular processes (58).

The cohesion within lipid rafts is suggested to be due to the lipid packing. The long and aliphatic tails of sphingolipids or phospholipids are assumed to snuggle to the smooth α-side of the sterol ring system (59). By doing so, larger headgroups of sphingolipids can provide an additional hydrophobic shield to cholesterol (60).

Clustering of lipid raft components induces larger, longer-lived structures, which are more accessible to investigation than the transient nanostructures observed in unperturbed cells (61). Clustering can be induced physiologically e.g. by (polyvalent) ligand binding to receptors, or experimentally, e.g. by binding of polyclonal antibodies to plasma membrane components. In addition, stabilization of membranes enriched in lipid raft components can also be achieved by extraction with mild, non-ionic detergents. The detergent extraction procedure is thought to preferentially extract lipids of more disordered membrane regions thus inducing the coalescence of small ordered regions, which then resist further extraction and thus are called detergent-resistant membranes or DRMs. Common procedures for this purpose are based on detergents like TX-100 (62), CHAPS (63), Lubrol WX (64), Brij 96 (65) or Brij 98 (51, 66, 67), and involve extraction regimens at 4^oC or 37^oC for short or long periods.

However, it is unclear to what extent the DRMs formed by these procedures resemble physiological lipid rafts (68). Nevertheless, the heterogeneity of lipid rafts and their association with distinct cellular processes is reflected at least in part by selective detergent extraction protocols that yield distinct types of DRMs that appear to correspond closely to different types of lipid rafts in a cell (67, 69).

2. Working hypothesis

9

2. Working hypothesis

We hypothesized that there is an early interaction between BCR and Ii-MHC II induced by the clustering of BCR by polyvalent antigen at the cell surface.

BCR was found in DRMs and by implication in lipid rafts after crosslinking by polyvalent antigen (70-72). In addition, MHC II and Ii-MHC II were demonstrated to distribute into lipid rafts during their intracellular journey and to exhibit a high degree of association with lipid rafts at the cell surface (51, 73, 74).

Clustering by polyvalent antigen might drive the BCR to the same membrane domain that is already occupied by Ii-MHC II and thus may lead to an early, membrane domain-guided convergence of clustered BCR and Ii-MHC II complexes. By doing so, lipid rafts might serve as sorting platforms for these components, which are supposed to traffic together to MHC II peptide loading compartments following BCR activation.

Fig. 2: Hypothetical lipid raft-guided interplay between Ii-MHC II and BCR upon polyvalent clustering of BCR.

3. Synopsis of results

3. Synopsis of results

This study demonstrates an early lipid raft-guided interplay between BCR and Ii-MHC II already at the cell surface. To date it is the first report about such an interaction. However, this teamwork is dependent on clustering both molecules individually by using polyclonal antibodies. In addition, the physiological ligand of Ii, Macrophage migration inhibitory factor (MIF), also triggered coalescence of BCR and Ii-MHC II upon its clustering by polyclonal antibodies. Contrary to the expectations, clustering of only BCR did not lead to a co- recruitment of Ii and vice versa. By performing a mild detergent extraction procedure and flotation analysis we demonstrated that clustered BCR and Ii were found in DRMs implicating their raft association. The assumption of an interaction between BCR and Ii could be verified by isolating Ii-containing DRMs that also contained clustered BCR. As to be expected for molecules present on the same DRM, the interaction between both components was highly susceptible to prolonged detergent exposure. Finally, we demonstrated that interaction of BCR and Ii-MHC II complexes induced by individual clustering led to augmented and prolonged intracellular tyrosine phosphorylation compared to BCR-mediated signaling only.

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5. Discussion

5.1 Weak association of clustered BCR with detergent resistant membranes (DRMs)

Earlier studies from several groups provided data demonstrating that antigen-mediated clustering triggered the BCR’s translocation into lipid rafts (17, 70, 75-77). These studies based their conclusion mostly on results from short-term extraction with 1 % TX-100 and DRM flotation in the absence of detergent. In all of the cases, the degree of flotation of the BCR after antibody or antigen-mediated crosslinking was only low. Indeed, in one work even 1 % digitonin was used for extraction of BCR-containing DRMs- a detergent usually employed to solubilize conventional, cholesterol and sphingolipid-rich DRMs (13). Thus the type of DRM the BCR is recruited to after antibody-mediated crosslinking appears to be highly unusual. It is also remarkable that no DRM association has been detected in the absence of crosslinking antibodies or antigen by any of these studies. These results reflect on our difficulty to efficiently isolate clustered BCR by conventional detergent extraction methods.

Only a very mild detergent extraction protocol developed by He's laboratory for the isolation of T-cell receptor-containing DRMs proved to be well suited for this task (67). There is a striking similarity in the extraction results between the BCR and the TCR: in an unligated form, both receptors distribute only very partially to DRMs, whereas upon ligation of the receptors with antibodies or the cognate ligand, the fraction of receptors in DRMs strongly increases (67). Although an increase in DRM association after clustering has been observed with many membrane proteins (69, 78), the magnitude of this effect is far below the one observed for BCR or TCR. It is interesting that the BCR and the TCR, which are both known to be expressed in myriads of variants differing in antigen or MHC-peptide specificity, show such a weak, tunable DRM association. Both types of receptors need to be able to discriminate between closely related ligand structures with exquisite fidelity. DRM- association and by implication membrane raft association may help to overcome a threshold for activation of these receptors and the generation of downstream signals. Indeed, for B- cells a requirement for membrane rafts in the very early steps of receptor activation has been demonstrated (21). This appears to pertain to T-cells as well, although an involvement of lipid rafts in the early steps of T-cell activation is still disputed (79-81). Nevertheless, the work by Drevot et al. showed that the TCR signal initiation machinery (comprising TCR–CD3

5. Discussion

complex, Lck and ZAP-70 kinases and CD4) was resistant to detergent extraction suggesting a role for lipid rafts in the early steps of TCR-mediated T-cell activation (67).

5.2 Clustering of invariant chain: not only an artificial approach

Earlier studies revealed that the physiological mechanism that triggers BCR activation was thought to be physically crosslinking by soluble multivalent antigen (13). Later on, evidence was provided that physically crosslinking is not a prerequisite for BCR signaling triggered by a membrane anchored antigen (14). This is in agreement with observations that, indeed, B- cells encounter and respond to antigen both in solution and on so-called antigen-decorated cells, like conventional antigen presenting cells (APCs) (15, 16) or follicular dendritic cells.

Moreover, both mono- and multivalent antigen incorporated into fluid bilayers were demonstrated to induce microcluster formation and signaling of cognate BCR (82).

Our findings suggest that molecules capable of clustering Ii during antigen detection by the BCR influence the early events of BCR induced B-cell activation by bringing both BCR and Ii in close proximity. Macrophage migration inhibitory factor (MIF) has been shown to serve as a physiological ligand of invariant chain (83, 84) in addition to the chemokine receptors CXCR2 and CXCR4 (85). There is only a very small fraction of Ii present at the cell surface (41, 86). It has been assumed that surface Ii is modified by a proteoglycan (87), associated with CD44 (88,89), CXCR2 (85) and CXCR4(90). However, there are no quantitative data on what fraction of surface Ii is complexed by these or other molecules. Earlier work has revealed that a substantial amount of newly synthesized Ii-MHC II complexes is cycling back and forth between the plasma membrane and early endosomal compartments before its retrieval to later endocytic compartments (41). As shown in this work (Fig. 1), only about 3 % of surface invariant chain was present as free invariant chain trimer in NHHT-39 B-cells, whereas the remaining material was in fact complexed to MHC II. We neither found evidence for substantial amounts of other association products of invariant chain at the plasma membrane, nor did we detect evidence for a high molecular weight form of Ii by immunoprecipitation of velocity gradient fractions (Fig. 1). Although this analysis does not rule out that complexes of surface Ii with other proteins or high molecular weight modification products of Ii exist in NHHT-39 cells, it shows that such complexes can only be present in very small quantities, if at all. The most likely binding partners for MIF are

5. Discussion

29 therefore either MHC II-Ii-complexes or the small quantity of free Ii that was detected at the surface of NHHT-39 cells (Fig. 1)

The mere binding of MIF did not lead to its recruitment to BCR clusters. Instead, independent clustering of both, BCR and MIF (bound to Ii) was required (90) to induce co- localization of the surface of NHHT-39 cells. This result was similar to what we found for the receptor of MIF, Ii, when this molecule was clustered by antibodies (91). It was surprising that a trimeric ligand bound to its trimeric receptor did not trigger receptor oligomerization and subsequent co-clustering with patched BCR by itself. Possible explanations for this failure could be i) an excess of MIF in our experimental setting, ii) a possible 1:1 binding of a MIF trimer to a trimer of Ii or iii) an inability of Ii-MHC II to bind more than 1 MIF trimer. The binding site of MIF has been shown to be contained in a soluble fragment of Ii (Ii73-232) comprising the MHC II binding site and the C-terminal trimerization domain (83, 84). Binding in this region of Ii would be incompatible with an 1:1 stoichiometry, unless it occurred right on the top end of the Ii molecule. This, however, appears to be unlikely, since recent investigations showed that partial, soluble MHC II molecules consisting of linked α1, β1 subunits and antigenic peptide interfered with the interaction of MIF and Ii (92, 93). If confirmed for intact MHC II, these results would suggest that binding of MIF to invariant chain is on the lateral side of the Ii trimer (close to the MHC II binding site) and therefore should occur in a stoichiometry of more than one MIF trimer per trimer of Ii. Their results furthermore suggest that nonameric Ii-MHC II complexes do not interact with MIF. Binding of MIF to Ii should therefore to be expected to occur either on the small population of MHC II-free Ii trimers (about 3 % of the surface Ii molecules, see Fig. 1) or to Ii-MHC II complexes that contain fewer than three MHC II molecules per Ii trimer. Such complexes could offer 1-2 MIF binding sites per Ii trimer, while the rest would be blocked by bound MHC II. Evidence for such substoichiometric Ii-MHC II complexes is still weak (40, 94) but also see comments by Lindner and Cresswell (Plos ONE, 2011). Our finding that the two Ii-MHC II species on the surface of NHHT-39 cells sedimented much slower than expected for an Ii-MHC II nonamer (Fig. 1) support a scenario with unsaturated Ii-MHC II complexes offering free binding sites for MIF. Further work is required to clarify the structure of Ii-MHC II complexes on the surface of B-cells and to elucidate their role as receptors of MIF.

By contrast to clustering of Ii-MHC II, the oligomerization of the BCR by polyvalent antigen is a well-studied process that bears physiological relevance for B-cell activation by soluble

5. Discussion

antigens (13, 14). Recent evidence, however, reveals that other pathways for the delivery of antigens to B-cells exist in vivo. They involve the display of non-degraded antigen on the surface of DCs (16), macrophages (15) or follicular dendritic cells (95). On their itinerary through the lymphatic system, B-cells pass these so-called antigen-decorated cells and become activated once they detect their cognate antigen on the surface of the displaying cell (18). Extensive work by the laboratories of Batista/Neumann and Pierce has shown that the recognition of membrane-bound monomeric antigen also results in BCR clustering, subsequent triggering of the signaling cascade and finally in endocytosis (reviewed in (18)).

We therefore asked ourselves whether a physiologic setting like this could induce the oligomerization of Ii-MHC II and its co-clustering with BCR patches. In support of such a scenario, the chemokine receptors CXCR2 and CXCR4 have recently been described as alternative receptors for MIF (85), which may function as display receptors for MIF on the surface of antigen-decorated cells (ADCs). In fact, cell-bound MIF has been shown to be able to activate CXCR2 and CXCR4-expressing cells in trans (85). Since the chemokine receptors appear to bind to a different part of the MIF molecule than Ii (90) it is conceivable that chemokine receptor-bound MIF on an ADC might interact with Ii on a B-cell during an episode of cell contact. In analogy to the BCR, such an event might trigger the clustering of Ii and this in turn should lead to the coalescence of the BCR and Ii, if also the BCR had been clustered by cell-bound antigen (as demonstrated by Batista and Neuberger (14)). Our considerations are summarized in a hypothetical model depicted below (Fig. 3):

5. Discussion

31 Fig. 3: Hypothetical model for the simultaneous presentation of membrane-bound antigen and MIF by an

antigen-decorated cell (ADC) to a B-cell. Recognition of antigen by the B-cell receptor (BCR) and of MIF by an (substoichiometric) Ii-MHC II complex leads to microcluster formation and convergence in a common lipid raft in the B-cell membrane. Co-endocytosis and subsequent co-targeting to MHC-II peptide loading compartments as well as augmented signaling facilitate the processing and the presentation of cognate antigen by the B-cell.

5.3 A novel role for the pro-inflammatory cytokine MIF (Macrophage migration inhibitory factor)

MIF is a pro-inflammatory cytokine that is produced by a wide variety of cell types including monocytes/macrophages, B- and T-lymphocytes as well as non-immunological cells like endocrine, endothelial and epithelial cells (96). In its active form it is a homotrimeric molecule that is functionally and topologically homologous to the D-dopachrome tautomerase (DDT or MIF 2) (97, 98). MIF 1 has been reported to be involved in infectious diseases, cancer, autoimmune and metabolic disorders (reviewed in (96, 99)). A wide variety

5. Discussion

of pro-inflammatory, anti-apoptotic, proliferative and also some chemokine-like functions have been ascribed to this molecule. Apart from its name-giving role in arresting macrophage migration (100, 101), one of the first reported physiologic effects of MIF was the counter-regulation of glucocorticoid suppression of immune cell responses (102). It was revealed that low concentrations of glucocorticoids induced MIF production by macrophages and helped to override glucocorticoid-mediated inhibition of inflammatory cytokine secretion in vitro and in vivo. In another report antisense RNA was used to generate MIF- deficient macrophages and to show that toll-like receptor-4 expression was dependent on MIF in this cell type. It established a critical function for MIF at the interface between innate and acquired immunity (103, 104). Further support for a pro-inflammatory function of MIF was provided by the finding that it inhibits p53-mediated apoptosis and thereby prolongs the survival of activated immune effector cells (103, 104). In addition, chemokine-like functions of MIF have been observed in inflammatory diseases and atherogenesis (85, 105).

Last not least, effects of MIF on adaptive immune responses have been reported, centering on its involvement in T_H2-dependent immune reactions (106-111). An initial study showed that antibody-mediated depletion of MIF in vitro and in vivo led to diminished antigen-driven T-cell activation and antibody production (106). With the availability of MIF knockout mice, detailed analyses in models for T_H2 type immune reactions were performed. In all models, significant reductions in T_H2-controlled immune responses were observed with MIF knock- out mice, leading to reduced atopic reactions, reduced antibody production or increased infection (107, 109-112). These studies also showed that MIF functions at multiple levels in T_H2 responses and affects multiple cell types likes macrophages, T_H2 cells B-cells. Apart from its effect on signal transduction via Ii and supposedly associated proteins like CD44 (113), no molecular mechanism has been proposed, yet, by which MIF could enhance B-cell activation and BCR-triggered antigen processing and presentation on MHC II. Our data and the hypothetical model derived from them (Fig. 3) attempts to fill this gap and could serve as a basis for future investigation.

6. Summary

I

6. Summary

The B-cell receptor (BCR) transiently associates with lipid rafts upon recognition its cognate antigen. Encountering antigen induces BCR clustering and targeting to endocytic processing compartments, which are also accessed by invariant chain-MHC II complexes (Ii-MHC II). In addition, a small portion of total Ii-MHC II is present at the cell surface and described to reside in lipid rafts as well. This study was performed to investigate whether there is a lipid raft-guided interaction between BCR and Ii-MHC II that already occurs at the cell surface.

First, confocal laser scanning immunofluorescence microscopy was used to investigate the distribution of BCR and Ii-MHC II on the surface of B lymphoma cells after antibody-mediated clustering. It could be shown that, indeed, antibody-induced polyvalent clustering of BCR and Ii- MHC II lead to colocalization of both molecules at the cell surface. However, colocalization required clustering of both type of molecules. Clustering of BCR only did not redistribute Ii-MHC II to the BCR or vice versa. Macrophage migration inhibitory factor (MIF) -the physiological ligand of Ii- was found to induce colocalization of Ii-MHC II and BCR after additional antibody mediated oligomerization. The fact that Ii-MHC II and the only other protein detected to be co-clustering with BCR in this study, peptide-loaded MHC II, were reported to be lipid raft-associated suggested a lipid raft guided convergence of BCR and Ii-MHC II. This convergence was independent of F-actin and led to subsequent co-endocytosis.

Our microscopic approach was complemented by a biochemical analysis of the BCR and Ii involving detergent extraction and flotation into density gradients. The flotation analysis revealed a low tendency of clustered BCR to distribute to detergent-resistant membranes (DRMs). However, by using a rapid Brij 98 extraction method, BCR was detected in the detergent-resistant fractions, where it co-floated with Ii suggesting a lipid raft mediated interaction. This finding was confirmed by an immunoisolation experiment, in which the isolation of Ii-containing DRMs led to the co-purification of clustered BCR.

Finally, downstream signaling - the other main effect of BCR activation besides endocytosis - was found to be enhanced by coalescence with Ii as shown by increased tyrosine phosphorylation.

Our data suggest a model, in which cell-bound antigen and MIF trigger the coalescence and the endocytic co-sorting of BCR and Ii-MHC II already at the cell surface. This event might affect BCR- mediated processes like antigen processing and presentation.

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9. Erklärung nach § 2 Abs. 2 Nrn. 6 und 7 der Promotionsordnung

IV

9. Erklärung nach § 2 Abs. 2 Nrn. 6 und 7 der Promotionsordnung

Ich erkläre, dass ich die der Medizinischen Hochschule Hannover zur Promotion eingereichte Dissertation mit dem Titel

Coalescence of

B-cell receptor and invariant chain-MHC II in a raft-like membrane domain

im Institut für Zellbiologie im Zentrum Anatomie unter Betreuung von Prof. Dr. rer. nat. E.

Ungewickell sowie Dr. rer. nat. R. Lindner im Rahmen des StrucMed-Programms zur strukturierten Doktorandenausbildung ohne sonstige Hilfe durchgeführt und bei der Abfassung der Dissertation keine anderen als die dort aufgeführten Hilfsmittel benutzt habe.

Die Gelegenheit zum vorliegenden Promotionsverfahren ist mir nicht kommerziell vermittelt worden. Insbesondere habe ich keine Organisation eingeschaltet, die gegen Entgelt Betreuerinnen und Betreuer für die Anfertigung von Dissertationen sucht oder die mir obliegenden Pflichten hinsichtlich der Prüfungsleistungen für mich ganz oder teilweise erledigt. Ich habe diese Dissertation bisher an keiner in- oder ausländischen Hochschule zur Promotion eingereicht. Weiterhin versichere ich, dass ich den beantragten Titel bisher noch nicht erworben habe.

Ergebnisse der Dissertation wurden in folgendem Publikationsorgan - Journal of Leukocyte Biology - veröffentlicht:

Hauser JT, Lindner R. Coalescence of B cell receptor and invariant chain MHC II in a raft-like membrane domain. J Leukoc Biol. 2014 Nov;96(5):843-55

Hannover, den 23.06.2015 ______________________________

10. Danksagungen

10. Danksagungen

Ich möchte mich sehr herzlich bedanken bei meinem Betreuer und Doktorvater Robert Lindner für die freundliche Überlassung des Themas und die stetig exzellente Unterstützung. Durch ihn mit seinem unermüdlichem Ehrgeiz für detailreiche und gute Wissenschaft, sein immer offenes Ohr für Fragen und Nöte und seine

ausgezeichnete Expertise ist diese Arbeit möglich geworden.

Des Weiteren möchte ich mich bei Prof. Ernst Ungewickell für die Möglichkeit bedanken, im Rahmen des StrucMed-Programms in seinem Institut diese Arbeit anzufertigen. Seine hilfreichen Anmerkungen und sein Interesse am Projekt haben immer wieder zum Fortschritt beigetragen.

Ebenso allen Mitarbeitern des Instituts Zellbiologie möchte ich danken für ihre Unterstützung, ihre technische Assistenz und die vielen schönen Stunden.

Besonderer Dank gilt an dieser Stelle Gudrun Daenecke.

Bedanken möchte ich mich auch recht herzlich bei allen Mitgliedern des

„Doktorandenzimmers“, welche meine Freunde geworden sind und mir immer wieder geholfen haben die vielen Rückschläge mit dem nötigen Lächeln zu sehen, um sich gemeinsam über die Fortschritte zu freuen. Besonders bedanken möchte ich mich hier bei Inga und Philip.

Zuletzt möchte ich mich bei meiner Familie bedanken, welche mir stets zur Seite steht und mich bedingungslos in allem bekräftigt, was ich tue. Denn der meiste Dank von allen gebührt meinen Eltern, welche mir ein gutes Elternhaus und das

Medizinstudium ermöglicht haben. Ohne euch wäre diese Arbeit nicht möglich gewesen. Danke!