Cell,

Vol.

67, 105-l

16, October

4, 1991,

Copyright

0 1991

by Cell

Press

Processed Antigen Binds to Newly Synthesized MHC Class II Molecules in Antigen-Specific B Lymphocytes Howard W. Davidson, Pamela A. Reid, Antonio Lanzavecchia, l and Cohn Watts Department of Biochemistry Medical Sciences institute University of Dundee Dundee DDl 4HN Scotland ‘Base1 Institute for Immunology Grenzacherstrasse 487 Postfach CH-4005 Base1 Switzerland

Summary We describe the direct detection of radiolabeled antigen fragments bound toclass II MHC moleculesfollowing immunoglobulin-mediated endocytosis and processing of native antigen in B lymphoblastoid cells. Tris-Tricine SDS gels revealed six distinct iodinated processing products that could be detected on class II MHC 1 hr after antigen endocytosis and persisted for at least 20 hr. These physiological processed antigenclass II complexes were remarkably stable, as judged by the fact that class II ab dimers, which remain associated in SDS, became labeled with the same set of processed peptides. Using a lectin-binding assay, we show that these physiological processing products bind to the newly maturing population of MHC molecules rather than binding to the preexisting cell surface population; in contrast, an exogenous peptide binds predominantly to the latter population. A direct T cellindependent assay for processed peptide-MHC complex formation should facilitate additional studies on the exogenous antigen processing pathway. Introduction T lymphocytes recognize peptides in association with either class II or class I molecules of the major histocompatibility complex (MHC). Earlier indirect evidence for this (reviewed in Unanue, 1984; Townsend and Bodmer, 1989) received direct support from the structural analysis of class I molecules (Bjorkman et al., 1987) as well as from the biochemical analysis of purified class II molecules (Buus et al., 1988; Demotz et al., 1989b) and, recently, class I molecules (Van Bleek and Nathenson, 1990; Rotzschke et al., 1990; Falk et al., 1991). These data reveal that the presumptive peptide products of antigen processing are intimately associated with MHC glycoproteins and raise a variety of questions about how and where these complexes are generated under physiological conditions. Current evidence suggests that the assembly of class I and class II MHC molecules into complexes with peptides takes place at different intracellular locations and that binding of peptides, at least to class I MHC molecules,

induces a stable conformation compatible with accumulation on the cell surface (Townsend et al., 1989, 1990; Shumacheretal., 1990). In thecaseofclassll MHCmolecules, initial peptide binding is thought to occur following processing of the invariant chain at some point along the endocytic pathway (Elliott et al., 1987; Blum and Cresswell, 1988; Roche and Cresswell, 1990a; Teyton et al., 1990; Neefjes et al., 1990; for reviews see Long, 1989; Koch et al., 1989; Yewdell and Bennick, 1990; Brodsky and Guagliardi, 1991; Braciale and Braciale, 1991). It seems that only a small proportion of exogenous or endogenous antigen is processed to yield MHC-peptide complexes, which has prevented the analysis of their formation at a biochemical level. Generally, T cells are used to detect the appearance of such complexes on the cell surface and, more recently, for the isolation and characterization of physiologically processed peptides. This approach has yielded important new information regarding the size, the heterogeneity, and in the case of naturally processed class I epitopes, the sequence of physiologically generated determinants (Demotz et al., 1989b; Van Bleek and Nathenson, 1990; Rotzschke et al., 1990; Falk et al., 1991). However, T cell assays cannot easily be adapted to probe the site of physiological antigen processing, assembly of peptide-MHC molecule complexes, and transport to the cell surface. These events could be analyzed directly if loading of MHC molecules with physiologically processed antigen could be measured as a biochemical event. The organization of the class II-restricted antigen processing pathway has been analyzed by biochemical studies on antigen endocytosis and processing (Watts and Davidson, 1988; reviewed in Watts et al., 1990) by analysis of class II biosynthesis and trafficking (Neefjes et al., 1990; Lotteau et al., 1990; Bakke and Dobberstein, 1990; reviewed in Cresswell et al., 1990), and by morphological studies(Guagliardiet al., 1990; Petersetal., 1991). Recent studies show that class II molecules diverge from class I molecules, probably in the trans-Golgi network, and reach compartments accessible by endocytic markers (Cresswell, 1985; Neefjes et al., 1990; Peters et al., 1991). Although earlier studies indicated that these newly synthesized class II molecules are targeted to early endosomes (Guagliardi et al., 1990), more recent work supports the idea that they are localized in a distinct late endosome population, primarily in communication with the Golgi network and lysosomes (Peters et al., 1991). However, the site of formation of peptide-MHC molecule complexes remains elusive. Another issue concerns the possible reuse of mature class II molecules: studies using peptide epitopes that do not require additional processing have revealed suprisingly short half-lives for some peptide-class II complexes in living cells, raising the possibility that a single class II molecule might regenerate its binding site and exchange peptides, perhaps during recycling (Harding et al., 1989; Adorini et al., 1989; but see also Lee and Watts, 1990).

Cell 106

Ant!-class

P

62 25

other studies show that presentation of native antigen is sensitive to drugs, such as brefeldin A and cycloheximide, that presumably do not interfere with the cycling population (St.-Pierre and Watts, 1990; Adorini et al., 1990). Again, if complex formation could be detected at the biochemical level, the origin of the class II molecules involved in binding newly processed peptides might be directly determined. To analyze the cellular biochemistry of class Il-restricted antigen processing, we have used a system in which the endocytosis and delivery for processing of antigen molecules is receptor mediated via membrane immunoglobulin (Ig). Using human lymphoblastoid cells specific for tetanus toxin (Lanzavecchia, 1985) we have shown that monovalent antigen is efficiently taken up through coated pits and delivered some 1 O-20 min later, still bound to lg, for processing. Distinct lg-associated antigen fragments arise in 6 cell clones with different epitope specificity, and we have been able to detect distinct fragments in both endosomal and lysosomal compartments (Watts et al., 1989; Davidson and Watts, 1989; Davidson et al., 1990). In this system, processed antigen-MHC molecule complexes can first be detected on the cell surface approximately 1 hr after initial antigen uptake, as measured by Ca’+ mobilization in antigen-specific T cell clones (Roosnek et al., 1988). We have now detected the loading of processed antigen fragments onto class II molecules at the biochemical level, and we have developed an assay to show that these fragments become bound to the newly maturing population of class II molecules. In contrast, an exogenously added peptide that does not require processing binds primarily to the cell surface/recycling population. Results Loading of Class II MHC Molecules with Processed Antigen To detect loading of class II MHC molecules with radioio-

Figure 1. Kinetics MHC-Associated Fragments

II

*

*

20

4 4

of Appearance and Ig-Associated

of Class II Antigen

A46 cells were incubated with ‘251-labeled antigen at 0°C and washed, and aliquots (1 O’cells) were incubated at 4 x lo6 cells per ml (O-6 hr timepoints)or 106cellsperml(20 hrtimepoint), as described in Experimental Procedures. After preclearing with protein A-Sepharose to remove intact lg, class II MHC and membrane lg fragments were recovered by sequential precipitation with DA6.231 and QE 11 (anti-K) antibodies and electrophoresed on 16.5% TrisTricine gels. (Note that most Ig-associated antigen fraqments are bound to cleaved, non-prot&in A-precipitable forms of lg; Davidson et al., 1990.) Two previously observed Ig-associated antigen fragments are indicated (‘) on the left panel, while arrows to the right indicate six consistently observed antigen fragments, which specifically immunoprecipitate with the DA6.231 MAb.

dinated antigen fragments, we used clone A46 cells, because this clone expresses the highest number of antigen-binding sites among the available clones (-3 x lo4 per cell). The cells were incubated at 0°C with radioiodinated tetanus toxin, washed, and then shifted to 37% for different times to allow endocytosis and processing of antigen. The 150 kd tetanus toxin molecule becomes progressively degraded, and distinct clone-specific and Ig-associated fragments appear with apparent molecular weights of 15 kd and 4 kd (Davidson and Watts, 1989; Figure 1). After various incubation times, the class II molecules were also immunoprecipitated with the 6 chain-specific DA6.231 antibody (Guy et al., 1982). Analysis of these immunoprecipitates on 16.5% Tris-Tricine SDS gels revealed a set of six fragments (see arrows in Figure 1) that appeared in a time-dependent fashion. Unlike the Ig-associated fragments characteristic of this cell clone (Davidson and Watts, 1989 and Figure l), the fragments were still persistent in the cells at the longest time point (20 hr; Figure 1). The fragments could not be precipitated with anti-class I antibodies, and their appearance on class II molecules was absolutely dependent on membrane lgmediated endocytosis, since they were not observed in another lymphoblastoid cell line (EDR) that is not specific for tetanus toxin (Figure 2). The appearance of class II MHC-associated antigen fragments was dependent on antigen metabolism following antigen uptake, since their appearance was reduced by 40% at 25% and eliminated at 18’C, although a reduced level of endocytosis still occurs at this temperature (Dunn et al., 1980; Davidson and Watts, 1989; Harding and Unanue, 1990). Chloroquine effectively blocks class II-restricted antigen presentation (Ziegler and Unanue, 1981) and in our study also virtually eliminated the appearance of the labeled fragments on class II MHC molecules (Figure 2).-Quantitation of the radiolabel recovered in individual fragments showed that brefeldin A inhibited the loading of class II molecules by 450/o-50%; in the presence of the thiol protease inhibitor leupeptin, on the other

Processed

Antigen

Sindrng

to Class

II MHC

107

11

6.2 2.5

OllS

A46

A46

rnAb

DA6 231

-

A46

EDR

VW32

DA6 231

hand, there was not only a marked reduction in the amount but, interestingly, a clear change in the mobility of some of the fragments recovered (Figure 2). We performed control experiments to assess the possibility that these fragments became bound not during intracellular antigen processing but following release either into the incubation medium or into the cell lysate. Fresh cells were incubated for 5 hr in medium previously “conditioned” by an equal number of cells pulsed with radiolabeled antigen for 5 hr. While class II molecules isolated from the pulsed cells displayed the expected set of antigen fragments (Figure 2, lane 1 l), those immunoprecipitated from the second set of cells did not (lane 12) ruling out a release and recapture mechanism. Two additional cell aliquots, one of which had been pulsed with radiolabeled antigen, were disrupted mechanically and a membrane and soluble fraction prepared from the postnuclear supernatant (see Experimental Procedures). The high speed supernatant from the pulsed cells was mixed with the membrane fraction prepared from the unpulsed cells and vice versa. After addition of detergent, class II molecules were precipitated from both mixtures. Only the membranes prepared from the pulsed cells carried class II molecules labeled with antigen fragments. Although the high speed supernatant contained, in different experiments, between 8% and 12% of the radioactivity recovered in the postnuclear supernatant, it could not provide a source of peptides for labeling class II molecules solubilized from unpulsed membranes (Figure 2, lanes 13 and 14). We conclude that binding of these fragments occurs during the course of physiological antigen processing. In spite of several preclearing steps, there was always a variable amount of undegraded (150 kd) antigen precipi-

12

13

14

Figure 2. Conditions for Loading Molecules with Processed Peptides

of Class

II

A46 or EDR cells were preincubated at 0% with ‘Z51-labeled antigen, washed, and chased at 37% for 5.5 hr at 37% or the temperature indicated. Prior to antigen binding, some aliquots were preincubated with IO uglml brefeldin A for 30 min or with 500 pglml leupeptin for 90 min and then, after antigen binding, in the same concentrations of either drug during the chase. Chforoquine was added at 0.5 mM without preincubation. The cells were collected and class II molecules precipitated with the DA6.231 antibody as described in Experimental Procedures. Control precipitations had no primary antibody (lane 2) or 5 ug of purified W6/32 to precipitate class I MHC molecules (lane 3). Class II molecules were also precipitated (lane 12) from cells that had been incubated in growth medium previously conditioned for 5.5 hr by antigen-pulsed cells (lane 11). In addition, a membrane fraction (lane 13) was isolated from nonpulsed cells, which was then mixed with a high speed supernatant prepared from pulsed cells whose own membrane fraction is analyzed in lane 14 (see Experimental Procedures for details). Note that in each lane 1.7 x 10’ cells are analyzed. Selected bands from lanes 5-10 were quantitated on a Molecular Dynamics Phosphorimager.

tated along with the fragments (Figures 1 and 2). In fact, this represents a very small fraction of the total intact antigen remaining in the cells (~0.5%) and probably represents an unavoidable background, since it was also observed to some extent in control immunoprecipitations (Figure 2). However, it is possible that this represents intact antigen specifically bound to class II in an unfolded conformation, as has been described in other systems (Lee et al., 1988; Sette et al., 1989). Loading of SDS-Stable Class II ap Complexes with Processed Peptides The antigen fragments recovered on class II molecules after 5 hr of processing constituted about 0.1% of the radioactivity initially bound to membrane lg, and the quite long autoradiographic exposures (~2 weeks) required to visualize these fragments limited the usefulness of the assay. Earlier studies showed that a proportion of class II a and f3 chains run together as a complex on SDS gels, provided that the sample is not heated (Springer et al., 1977; Cresswell, 1977). We reasoned that SDS-stable ab complexes might retain physiologically processed peptides and would thus be concentrated in one position on the gel, reducing the time required to see a signal. This would also provide a rigorous way of analyzing the mode of association of these peptides with class II, since material bound nonspecifically might be expected to be eluted in SDS. To test this, class II molecules were isolated either from surface-iodinated cells or from cells that had been pulsed with radiolabeled antigen for 5 hr. The immunoprecipitates were eluted into SDS sample buffer at room temperature (m23”C) or at 100°C and analyzed on standard 15% SDS gels. As shown in Figure 3a, analysis of surface-

Cell 108

(b)

(4 Cell surface 125,

1

2 15%Tw/Glycine

Figure

3. SDS-Stable

EXClS&np

complexi10o”C

,25 I Antigen

+DTT -DTT

34

1 gel

a5 Complexes

2

16.5 % Triflriclne

Carry

Processed

f

3 gel

Peptides

(a) A46 cells were either surface labeled with 1251-labeled sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate (see Experimental ProceY-antigen for 5 hr at 37%. After dures) or pulsed with Ig-bound preclearing with Pansorbin followed by OEll MAb (to remove Ig-associated antigen), class II MHC immunopreclpitates were eluted either at 23OC (lanes 1 and 3) or at 95% (lanes 2 and 4) and run on a 15% standard SDS gel. (b) Cells (4.5 x 10’) were pulsed with Ig-bound ‘251-labeled antigen for 5 hr, and class II MHC molecules were lmmunoprecipitated from ~2.5 x 10’ cells, eluted in SDS at 23%, and run on a 15% gel as in (a). The a5 complexes were excised and processed as in Experimental Procedures, and the gel piece was loaded directly onto a 16.5% TrisTricine gel (lane 3) adjacent to class II immunoprecipitates from the same batch of cells eluted at 95% in reducing (lane 1) and nonreduc~107cellseach). In lane3, approxing(lane2)SDSsample buffer(from lmately 1200 cpm from the a5 complex were loaded. In lanes 1 and 2, approximately 8.000 cpm were loaded, but note that a considerable proportion of this radioactivity was in coprecipitated intact antigen.

iodinated cells confirmed that a proportion (40%) of the a and 6 chains fan as acomplex (M, = ~63 kd) and dissociated into a and f3 chains upon heating in SDS (Figure 3a, lanes 1 and 2). Class II MHC immunoprecipitated from 1251-labeled antigen-pulsed cells also showed a labeled band, which comigrated with the surface-iodinated class II afi complex (Figure 3a, lane 3). This band also disappeared when the sample was heated; however, instead of giving rise to labeled a and 6 chains, the radioactivity present in this complex now ran mostly at the dye front, consistent with it being in antigen fragments of an apparent molecular weight less than 12 kd (Figure 3a, lanes 3 and 4). We then compared the radiolabeled material in the SDS-stable up complex with the fragments displayed on Tris-Tricine gels (Figures 1 and 2). Class II up complexes, labeled during antigen processing, were excised from standard 15% gels, equilibrated in Tris-Tricine-SDS sample buffer and heated to 100°C for 5 min. The gel slices were then reelectrophoresed on a Tricine gel in a lane

parallel to class II immunoprecipitates heated to 100°C in the same sample buffer and run under reducing and nonreducing conditions. As shown in Figure 3b, the complex broke down to reveal six distinct fragments very similar to those displayed when the immunoprecipitates were directly analyzed (arrows in Figures 1 and 3b), except that the intact antigen and some other high molecular weight antigen fragments were, as expected, missing from the profile (Figure 3b, lane 3). Note that equilibration and reelectrophoresis of the gel pieces cause some distortion of the electrophoretic separation in this gel lane, such that the most mobile fragment is not well resolved. As judged by the time course of their appearance (Figure l), their persistence in the cells (Figure l), and most importantly, the fact that their association with class II MHC molecules is remarkably stable even in SDS (Figure 3) we conclude that these fragments represent authentic products of tetanus toxin processing that become associated with the peptide-binding sites of class II molecules. Using the appearance of the labeled a6 complex as an assay for loading of class II molecules with processed peptides, we compared the kinetics of antigen degradation with the appearance of these labeled complexes. Labeled complexes could just be detected after 1 hr of processing and accumulated thereafter (Figure 4) consistent with the kinetics of T cell triggering measured previously in similar cells (Roosnek et al., 1988). Intact antigen is progressively degraded with a half-life of ~2.5 hr, while the Ig-associated 15 kd fragment we identified earlier in these cells (Davidson and Watts, 1989) reaches a maximum at ~2 hr and then declines. As expected, chloroquine inhibited the formation of SDS-stable class II complexes by ~55% after 2 hr and by m90% after 5 hr (Figure 4). Processed Antigen Is Loaded onto Newly Synthesized Class II Molecules Two populations of class II MHC molecules might be involved in binding newly processed antigenic peptides: newly synthesized molecules undergoing maturation prior to reaching the cell surface (Cresswell, 1985; Cresswell et al., 1990; Neefjes et al., 1990; Peters et al., 1991) and/ or mature cell surface class II molecules that might be cointernalized with antigen and targeted to compartments involved in antigen processing. Experiments designed to assess the capacity of preexisting versus newly synthesized class II molecules to present physiologically generated peptides have yielded somewhat contradictory results, and indeed the existence of a recycling population of surface class II molecules and the lifetime and exchangeability of peptide-MHC molecule complexes are currently controversial issues (see Discussion). We found that preincubation with cycloheximide for 4 hr prior to antigen pulsing or inclusion of brefeldin Aduring antigen pulsing partially inhibited the loading of class II molecules with processed peptides (Figure 2 and data not shown). To clarify the role of preexisting versus newly synthesized class fl molecules in the presentation of physiologically processed peptides, we took advantage of the biochemical assay described above for class II moleculepeptide complex formation and adopted a new strategy,

Processed 109

Antigen

Binding

to Class

II MHC

(a)

plus 0.5mM chloroquine

Time:

Hours

0

1

2

3

5

5/1OO”C

2

(b)

5

66 ai+ 3

2

3

Time

Figure

4. Kinetics

of a6 Complex

Labeling

4

5

5

(hours)

n

Class

II ap complex

0

15kD

Ig-bound

q

Intact

fragment

antigen

by Peptides

(a) A46 cells were loaded with ‘%labeled antigen, and aliquots (6 x lo6 cells) were incubated at 37% for the times indicated, after which class II MHC glycoproteins were immunoprecipitated and eluted in SDS at 23°C or at 100DC as described in Experimental Procedures. Chloroquine (0.5 mM) was added to some aliquots simultaneously with the start of the 37°C incubation. (b) The band corresponding to the a6 complex was excised and radioactivity quantitated by gamma counting. Aliquots of the whole cells (5 x 104) were electrophoresed on a parallel 15% gel (not shown), and the bands corresponding to the intact antigen (150 kd) and the Ig-associated 15 kd fragment stabilized in these cells (Figure 1 and Davidson and Watts, 1969) were excised and similarly quantitated. Values, which are expressed as a percentage of the maximum value, were: 150 kd antigen, 1.67 x 104cpm at t = 0; 15 kd fragment, 1.9 x 10’ cpm at t = 2 hr (both from -5 x lo4 cells) and a6 complex, 1.01 x i0” cpm at 5 hr (from 8 x 10’ cells).

whereby the preexisting cell surface population can be physically distinguished from those molecules that mature and arrive on the cell surface during the course of the experiment. Our approach is based on preliminary studies that showed that a relatively small proportion of cell surface class II molecules bind to the galactose-binding RCAfPO lectin (-30%) owing to the presence of terminal sialic acid residues, but, as expected, binding was increased considerably (mSO%) by treatment of the intact cells with neuraminidase. We reasoned that if the labeled peptides were becoming associated with the preexisting surface population of class II molecules, i.e., those on the

A46 cells + Neuraminidase

Elute SDS

1

at 23°C

Bind’25l antigen Wash, incubate 37”C, 5 hours or pulse with peptide

lmmunoprecipitate II from RCA,,, free fractions

v Lysate * Neuraminidase

4 Preclear, incubate with RCA,,,Agarose

in

4

-

Figure 5. A Protocol to Differentiate Synthesized Class II MHC Populations

between

bound

Preexisting

Class and

and

Newly

surface and accessible to neuraminidase before the onset of antigen processing, agreater proportion of labeled complexes should bind to the RCA,,o lectin when isolated from neuraminidase-treated cells relative to untreated cells. If, on the other hand, the processed peptides bind to newly maturing class II molecules, then prior neuraminidase treatment should make no difference, and in both cases the proportion bound to the lectin should be small. To test this, the experiment outlined in Figure 5 was performed. Neuraminidase-treated and control cells were incubated with radiolabeled antigen at O°C, washed, and then incubated at 37% for 5 hr. The cells were collected, and the cleared lysates were then incubated with RCAIPOagarose for 2 hr at 0%. As a control (see below), one set of cells was treated with neuraminidase at the lysate stage. Pilot experiments established the appropriate level of immobilized lectin to ensure adequate capacity for maximal binding of class II molecules from neuraminidase-treated cells (see Experimental Procedures). The unbound fraction was recovered, the agarose beads were washed, and galactose-terminated glycoproteins were specifically eluted. Class II MHC molecules were immunoprecipitated from both the unbound and the bound and eluted fractions, eluted in SDS sample buffer at room temperature, and run on 15% gels. In parallel, cells were suface iodinated, and neuraminidase-treated and control cells were lysed and

Cell 110

Neurammidase treatment Bound/Unbound to RCA 120

Pre Pvx. +

UB

+

Post proc. +

--+

--

+

UB

UBUB

Figure Newly

+

UB

4

a P

3

4

56

7

subjected to the same RCA,,,,-agarose chromatography procedure. The results of such an experiment are shown in Figure 6. The increase in RCAIPObinding on neuraminidase treatment of the cell surface was readily seen when surfaceiodinated cells were analyzed (lanes 7-10). Neuraminidase treatment increases the ratio of class II molecule binding to RCAIPO (bound/unbound) from 0.67 to 3.0, and this was maintained in cells reincubated for 5 hr at 37% (not shown), demonstrating that no detectable resialylation of class II molecules occurred; this is consistent with recent studies by Neefjes et al. (1990). Next, complexes radiolabeled with processed antigenic peptides were similarly analyzed and quantitated. The proportion of complexes bound to the RCA12,,-agarose was small and, strikingly, was virtually the same in neuraminidase-treated and untreated cells (bound/unbound ratio 0.28 and 0.22 respectively; Figure 6, lanes l-4). When the neuraminidase treatment was performed after the processing experiment, binding of the labeled complexes to RCAjPO occurred efficiently (bound/unbound ratio 2.5; Figure 6, lanes 5 and 6) demonstrating that labeled complexes isolated from the pretreated cells failed to bind because they were sialylated and not for other reasons. Since the failure of the processed peptide-labeled class II molecules to bind to the lectin cannot be explained by resialylation of the preexisting population during the experiment, and since a maximum of ~7% of this population could have been endocytosed at the time of neuraminidase treatment (Reid and Watts, 1990), we conclude that the physiological products of antigen processing identified in our assay are bound to new, sialylated molecules that become available during the time course of processing. Even when they were isolated from neuraminidase-pretreated cells, the class II MHC molecules labeled with processed peptides bound to RCAIPO less well than surfaceiodinated molecules isolated from untreated cells (bound/unbound ratios 0.28 versus 0.67; Figure 6). Therefore, by this assay we could not

6

9

10

6. Processed Synthesized

Antigen Peptides Class II Molecules

Bind

to

Neuraminidase-treated (Pre Proc.) and control (-)A46cells(aliquotsof 2.5 x 107)were loaded with lZSI-labeled antigen at O”C, washed, and incubated at 37OC for 5 hr. After preparing ly sates, one aliquot of previously untreated cells was incubated 10 hr at O°C with 100 mlJ/ml neuramininidase (Post Proc.). All lysates were adjusted to 5 mM EGTA, 5 mglml BSA, and 5 mM NaN3 prior to preclearing with Pansorbin. RCA,a-agarose was added, and class II MHC glycoproteins were precipitated from the unbound (U) and bound and eluted (6) fractions generated from each set of cells (lanes 1-6). The band corresponding to the a6 complex was excised from the dried gel and radioactivity quantitated by gamma counting. In parallel, A46 cells were surface iodinated by the lactoperoxidase method, and neuraminidase-treated and control cells were processed as described for the antigen-pulsed cells (lanes 7-10).

detect binding of physiologically processed peptides to the cell surface/cycling population. We then asked how an exogenous tetanus toxin-derived peptide epitope (tt 830-843), which binds with high affinity to the class II MHC molecule DRwll without additional processing (Demotz et al., 1989; O’Sullivan et al., 1990) would behave in this assay. Since the A46 cell clone does not express the DRwll haplotype, we used a different lymphoblastoid cell line, clone 11.3. Neuraminidasetreated and control cells were pulsed with 3 PM ‘251-labeled tt 830-843 at 37°C for 2 hr and then were processed in the same way as the antigen-pulsed cells. The results of a typical experiment are shown in Figure 7. The total recovery of ‘251-labeled tt 830-843 was virtually the same on neuraminidase-treated and control cells and corresponded to approximately 7.0 x lo3 molecules per cell, in good agreement with the data of Busch et al. (1990). However, in contrast to the class II molecules labeled with physiologically processed tetanus toxin peptides (Figure 6), the fraction of tt 830-843-labeled class II molecules binding to the lectin was greater on neuraminidase-treated cells (Figure 7) demonstrating binding to a preexisting class II population. Neuraminidase treatment of peptide-labeled class II molecules after cell lysis resulted in somewhat increased binding to RCA,*,, (Figure 7) suggesting that some peptide may bind to class II molecules that were not accessible to the neuraminidase treatment of intact cells. The similar behavior on RCAIPOof class II molecules labeled by surface iodination or by the exogenous peptide clearly contrasted with class II molecules that become labeled with processed antigen peptides (Table 1). Taken together, these results demonstrate that physiologically processed peptides and exogenous peptides bind to distinct populations of class II molecules. Discussion:

The results presented here describe a T cell-independent

Processed 111

Antigen

Binding

to Class

II MHC

Table 1. Physiologically Peptides Bind to Different

Pre-

NO%?

Neuraminidase Figure

7. Peptide

tt 830-843

Binds

Post-

treatment to Preexisting

Class

II Molecules

Approximately 10’1 I .3cells(DR3,wll)wereincubatedwith neuraminidase at O‘C, washed, and pulsed for 2 hr at 37°C with 5 Kg/ml ‘Wabeled tt 830-843 (specific activity -3 x lo4 cpmlng) in parallel with two untreated cell aliquots. The cells were washed, they were lysed in the presence of protease inhibitors, and one previously untreated aliquot was treated with 200 mU/ml neuraminidase for 3 hr at 0%. All samples were made 5 mM in EGTA, 5 mglml BSA, then precleared with protein A-Sepharose and glycoproteins fractionated on RCA,lO-agarose as described in Experimental Procedures. Class II molecules were immunoprecipitated with L243 antibody or DA6.231 antibodies. Peptide radioactivity in the washed immunoprecipitates is shown for a typical experiment.

measurement of processed antigen binding to class II MHC molecules during the course of antigen processing. The complexes were generated from a single pulse of Ig-bound antigen and could be directly detected in 5 x lo6 to 5 x 10’ cells. At least six distinct radioiodinated fragments of tetanus toxin were recovered on class II molecules. Remarkably, a proportion of these fragments remained stably bound to class II a8 dimers even in SDS, providing a stringent test of their mode of association with class II molecules and amplifying what is otherwise a weak signal from the individual labeled fragments. Only a small fraction of the input radiolabel (~0.1%) was recovered on class II molecules, which is probably to be expected since presumably only a small fraction of the radioiodinated peptides generated are able to bind, or survive long enough to bind, to class II MHC molecules. Moreover, those that can bind may face competition from unlabeled tetanus toxin peptides as well as from peptides derived from serum and, probably, from cellular proteins. It should be stressed that, while only 0.1% of bulk radiolabel was transferred from lg to class II, the transfer of specific processed regions of the molecule may well be much more efficient. However, this is difficult to quantitate at present, because we do not know the specific radioactivity of the individual fragments that are generated. Virtually all the iodinated processing products ran at the dye front of a standard 15% gel (Figure 3a), indicating a size range below about 12 kd. The Tris-Tricine system

Processed Peptides and MHC Class II Populations on RCAlzo

Exogenous

Bound/Unbound

Ratio

Class II Population Labeled by:

Preprocessing Neuraminidase Treatment

No Neuraminidase Treatment

Lectin Postprocessing Neuraminidase Treatment

Surface iodination

3.0

0.67

ND

Processed ‘*5-labeled tt peptides

0.28

0.22

2.5

‘251-labeled tt 830-844

1.7

0.66

3.2

Class II molecules were immunoprecipitated from neuraminidasetreated and control cells that had been either surface iodinated, pulsed with ‘251-labeled antigen, or pulsed with 1251-labeled tt 630-844. Prior to immunoprecipitation, each lysate was fractionated on RCAIPO. Quantitation of the experiments shown in Figures 6 and 7 was by direct counting of washed immunoprecipitates (surface-iodinated and peptide-pulsed cells) or, because of the presence of contaminating intact antigen, by excision or counting of the labelled a8 complex from the antigen-pulsed cells. Quantitation is expressed as the ratio of cpm bound/unbound to RCAjzO. ND, not determined.

(Shigger and von Jagow, 1987) resolves this material into approximately six distinct bands. Although we are only visualizing a subset of the bound processing products, the fact that we do not observe a smear of radiolabel suggests that a limited set of processing products are captured by class II molecules rather than a complex mixture of heterogenous peptides. The iodinated peptides recovered show considerable variation in apparent molecular weight (2-15 kd), but. because small peptides can run anomalously in SDS-Tricine gels (data not shown; see also Schumacher et al., 1991) we cannot be certain that the apparent molecular weights are accurate. Analysis of the labeled peptides by size-exclusion chromatography is made difficult by the low amounts of radioactivity recovered in each fragment, but preliminary characterization of material eluted from immunoprecipitated class II molecules and run under the conditions described by Buus et al. (1988) indicates a fairly broad peak of material of 1.5 10 kd (H. W. D. and C. W., unpublished data). Buus et al. (1988) found a similar size range for autologous peptides isolated from mouse I-Ad molecules, although a naturally processed hen egg lysozyme determinant isolated from I-Ed-expressing cells, while heterogenous, showed a relatively narrow apparent molecular weight range with a mean size of ~2 kd (Demotz et al., 1989b). Additional characterization of the processed peptides that we have detected will be necessary to establish their true size, but it seems likely that there is a greater size range for physiologically processed peptides bound to class II than for those bound to class I MHC, where the physiological processing products appear to be 8 or 9 residues in length (van Bleek and Nathenson 1990; Rotzschke et al., 1990; Schumacher et al., 1991; Falk et al., 1991).

Cell 112

Given the recent demonstration that peptides stabilize class I MHC conformation (Townsend et al., 1990; Schumacher et al., 1990) it is tempting to speculate that the processed peptides bound to class II a8 dimers might play an active role in stabilizing the dimer by making multiple contacts with both the a and 8 chains. Class II molecules isolated from a mutant lymphoblastoid cell line, which can present exogenous peptides normally but cannot present native antigen, dissociated completely in SDS at room temperature, consistent with the idea that processed peptides may stabilize class II molecules (Mellins et al., 1990). It would be interesting to know if exogenous peptidesstabilize class II molecules in these mutant cells, since some synthetic peptides can also remain stably bound to a8 complexes in SDS (Dornmair et al., 1989; Roche and Cresswell, 1990b; our unpublished data). We have utilized the biochemical assay for processed peptide-class II association to analyze the relative importance of newly synthesized versus preexisting class II molecules in binding processed peptides. Some studies have indicated that in living cells, class II-peptide complexes are quite labile, which has led to the suggestion that cell surface class II molecules may be able to regenerate their binding site and thus be occupied by different peptides at different times (Harding et al., 1989; Adorini et al., 1989). Since class II MHC endocytosis and recycling is a requirement for class II MHC molecules to be reused in this way, several recent studies have looked for such a cycle. Endocytosis and in some cases recycling have been detected by several groups using mouse B cells and B cell lines (Harding and Unanue, 1989; Salameroet al., 1990; Machy et al., 1990; Weber et al., 1990) and, in our own studies, using human B lymphoblastoid cells (Reid and Watts, 1990). However, other studies using similar human cell lines but different techniques have either not detected class II MHC endocytosis (Davis and Cresswell, 1990) or have observed endocytosis but not recycling (Neefjes et al., 1990). An additional prediction, if the preexisting population of class II molecules can present processed peptides, is that presentation should be relatively insensitive to drugs, such as brefeldin A and cycloheximide, that block the export of new class II molecules. However, no consistent picture has emerged: some studies report that exogenous (Harding et al., 1989; Nuchtern et al., 1990) and endogenous (Jaraquemada et al., 1990) antigen presentation via class II MHC molecules can be relatively insensitive to such drugs, while other studies (in some cases, studies of the same antigen presented by the same murine B cell line) found that presentation was sensitive (Adorini et al., 1990: St.-Pierre and Watts, 1990; Nadimi et al., 1991). We have directly measured the contribution of cell surface class II molecules versus maturing molecules in binding physiologically processed peptides. The assay avoids the use of drugs to block the biosynthetic pathway and instead utilizes a lectin chromatography step to distinguish preexisting from newly maturing molecules. The results show that physiologically processed peptides generated following Ig-mediated antigen uptake bind to the maturing population rather than the preexisting population of class

II molecules. However, using the same T cell-independent assay, an exogenous peptide bound primarily to the preexisting population. How do these results fit with our current understanding of the trafficking of class II molecules in relation to the kinetics of antigen uptake and processing? In our recent studies using the same cell clones used here, endocytosed class II molecules were returned to the cell surface very rapidly (t>,Z= 2-3 min; Reid and Watts, 1990), suggesting that they may not penetrate beyond the earliest endosome compartment. In contrast, processing of specific antigen is detectable only after a lag of 20-30 min (Davidson and Watts, 1989), and the appearance of processed antigen-MHC molecule complexes is detectable only after 45-80 min (Roosnek et al., 1988). Recent morphological studies also indicate that the major population of biosynthetic class II molecules is in a “late” endosomel lysosome site kinetically distant from the cell surface (Peters et al., 1991). Thus the sites of antigen processing and class II recycling are distinct, precluding, at least in these cells, the access of preexisting surface class II molecules to peptides generated during antigen processing. Recent studies measuring exogenous peptide binding to cells do not eliminate the possibility that newly synthesized class II molecules may be involved and that these molecules then appear on the cell surface during the incubation. The progressive increase in peptide binding seen over several hours at 37% (Busch et al., 1990) and the increased binding to living versus fixed cells (Ceppellini et al., 1989) would beconsistent with this. However, we found that the preexisting class II population clearly offers an alternative pathway for the binding of peptides supplied exogenously; these results extend to the biochemical level the many earlier studies on presentation of peptides by fixed cells (Shimonkevitz et al., 1983), as well as recent work demonstrating that peptide presentation to T cells is insensitive to cycloheximide and brefeldin A under conditions in which presentation of native antigen is sensitive (Adorini et al., 1990; St.-Pierre and Watts, 1990). At high concentrations and after prolonged incubation times, peptides will presumably bind to newly synthesized molecules; but using T cell activation as an assay, it might be hard to detect an effect of inhibitors of class II maturation if a large enough number of preexisting class II sites are present. The more rapid kinetics of peptide binding to fixed cells at mildly acidic pH (Jensen, 1990) or in living versus fixed cells (Ceppellini et al., 1989) is consistent with the idea that transient passage of surface class II molecules through an acidic compartment may enhance peptide binding. However, this remains to be established. We suggest that the relative physiological importance of preexisting versus newly synthesized class II molecules in presentation of exogenous antigens may depend on the extent of processing required by a given epitope. Preexisting cell surface class II molecules may only be physiologically important in a minority of situations, for example, when peptides are present in the external bathing medium and in thde cases when unfolded but otherwise intact molecules can form stimulatory complexes with class II molecules (Lee et al., 1988; Sette et al., 1989; Eisenlohr et al., 1988). In both cases, binding might be facilitated in

Processed 113

Antigen

Binding

to Class

II MHC

an acidic endosome compartment, perhaps in conjunction with some kind of peptide exchange mechanism (Adorini et al., 1989). However, for T cell epitopes requiring extensive processing, newly synthesized class II molecules are shown in this article to be the physiologically relevant pop ulation. At least in human lymphoblastoid cells, these appear to be the only class II molecules in later endosomes (Peters et al., 1991) where processed peptides are either generated or are delivered from other compartments (Harding et al., 1991). This model is consistent with a requirement for invariant chain for the presentation of some native antigens (Stockinger et al., 1989; Nadimi et al., 1991) and possibly the enhanced presentation of exogenous peptides in cells that lack it (Peterson and Miller, 1990) because the targeting and blocking functions (Roche and Cresswell, 1990a; Teyton et al., 1990; Bakke and Dobberstein, 1991) of the invariant chain will serve to maximize the loading of class II with peptides generated within the endocytic pathway, diminishing the sites available for exogenous peptides. The differential effects of brefeldin and cycloheximide observed in different studies may be less contradictory than they appear. T cell activation is dependent on a threshold of stimulatory complexes being reached that, as the biosynthetic pool declines after drug addition, may be achieved for some epitopes but not for others. Sensitivity to brefeldin can often be overcome by increasing the amount of antigen during the pulse (e.g., Nadimi et al., 1991). The biochemical readout described here measured the loading of newly synthesized class II molecules, yet this was only partially sensitive to brefeldin and cycloheximide. This underlines the point that adirect readout, which measures a product quantitatively, can complement cellular assays in which the threshold for a response may vary considerably from one T cell to another. In conclusion, we have developed a system in which processed antigen binding to newly synthesized class II molecules can be studied at a biochemical level independently of a T cell readout. This should facilitate additional analysis of the cellular biochemistry of the class ll-restricted antigen processing pathway. Experimental

Procedures

Antigen Tetanus toxin was a generous gift from Wellcome Biotech (Eeckenham, England) and was further purified as described and stored frozen at -70°C (Watts and Davidson, 1988). Ten microgram aliquots (20 ul) were thawed and iodinated with 10 ul of 100 mCi/ml Y (Amersham) by the lodogen method. The reaction was stopped by addition of 10 ~1 of saturated L-tyrosine followed by 10 ul of 0.5 M Nal. Unincorporated radioactivity was removed on a 6 ml column of Sephadex G-50 equilibrated in phosphate-buffered saline (PBS) containing 2 mglml bovine serum albumin (BSA), and peak fractions (void volume) were pooled and stored at 4°C. The protein was labeled to a specific activity of -5 x 10’ cpmlng. Peptides were iodinated by the same method, except that unincorporated iodine was removed on a 2 ml column of Sephadex GlO equilibrated in PBS. Cells and Antigen Processing A46 and other lymphoblastoid cells scribed (Lanzavecchia, 1985; Watts were collected and washed 2 times taining 5 mglml BSA and resuspended

were cultured as previously deand Davidson, 1988). The cells In Dulbecco’s PBS (DPBS) conat 2 x 1O’to 5 x 10’ ml in the

same buffer at 0°C. ‘251-labeled tetanus toxin was added to a final concentration of 1-2 Kg/ml, and the cells were incubated at O°C for 90 min. Unbound antigen was removed by washing in DPBS containing BSA, and the cells were resuspended at 4 x 1 06/ml in growth medium. Afler various times of incubation in a CO? incubator at 37”C, the cells and culture supernatants were collected for additional analysis. Cell Fractionation Cells, pulsed or unpulsed with radiolabeled antigen, were resuspended to 1.5 x 1 O’lml in 0.25 M sucrose, 5 mM Tris-Cl (pfi 8.0) 0.5 mM EDTA, plus the same protease inhibitor cocktail used for immunoprecipitation (Davidson and Watts, 1989) and broken by 20 passes through a steel ball bearing homogenizer (Balch et al., 1984; clearance, 14 urn). Nuclei and unbroken cells were removed by centrifugation at 2000 g for 10 min. This postnuclear supernatant was sonicated briefly to ensure lysis of large vesicular structures, and the membranes were then collected by centrifugation for 1 hr in the Beckman Airfuge (30 psi). The supernatant was carefully removed and the membrane pellets resuspended in 100 ul of immunoprecipitation lysis buffer (see below). Membranes from non-antigen-pulsed cells were mixed with the supernatant from pulsed cells and vice versa, and class II molecules were immunoprecipitated from each under the conditions described below. lmmunoprecipitation Cells were lysed at a density of 2 x 10’ to 3 x IO’ cells per ml in 50 mM Tris-HCI (pfi 7.5) 150 mM NaCI, 1% Triton X-100 (TBST) buffer containing a cocktail of protease inhibitors as described previously (Davidson and Watts, 1989). When neuraminidase digestion was performed on cell lysates, the addition of EGTA was delayed until after the digestion. The lysates were cleared first by centrifugation at 13,000 g for 10 min and then by two sequential incubations with 10% (v/v) washed Pansorbin (Calbiochem-Behring; 60 ullml lysate). In some experiments, an additional preclearing step was performed with 25 ul of Sepharose-coupled rabbit anti-mouse lg beads. This removed most of the unprocessed Ig-bound intact antigen and antigen fragments remaining in the cells. NaN3 and BSA (recrystallized) were generally added to the lysates to 5 mM and 2 mglml, respectively. Class II MHC molecules and IgG were precipitated with DA6.231 (f3 chain specific; Guy et al., 1982) and QE 11 (K chain specific; The Binding Site, England) monoclonal antibodies (MAbs), respectively, added as ascites (l-3 ullml lysate), and they were incubated at O°C for at least 2 hr. The immunoprecipitates were collected by the addition of affinity-purified rabbit anti-mouse lg serum (Serotec) coupled to cyanogen bromideactivated Sepharose at a concentration of 1 mglml (40 ul of a 50% suspension per ul MAb ascites). (Anti-human IgG reactivity was removed prior to coupling by passage through a column of human IgGagarose [Sigma].) The samples were incubated at least 2 hr at *lO“C with agitation, and the complexes were washed twice in TBST buffer and once in Tris-buffered saline alone. SDS sample buffer (2%) was added (1 v/v rabbit anti-mouse lg Sepharose) and either left at room temperature for ~20 min or heated to 100°C for 2 min and then collected by centrifugation through a small hole in the bottom of the tube. Neuraminidase Treatments and RCA12,-Agarose Chromatography Neuraminidase treatment of the cell surface was performed as described by Snider and Rogers (1985). Briefly, cells were resuspended to -7 x 10Yml in DPBS containing 5 mglml BSA (Sigma, recrystallized) and Vibrio cholerae neuraminidase (Calbiochem-Behring, 1 UI ml) added to a final level of 6 mu/lo6 cells. Where indicated, cell lysates (postprocessing) were adjusted to 1 mM CaCb and digested with 100-200 mu/ml neuraminidase for 3-10 hr at 0°C. Digestions were terminated by washing and/or by the addition of 5 mM EGTA. Pilot experiments to assess the capacity of RCAlso-agarose (Sigma) established that 10 ul of gel (~2 mglml protein) was required to bind the maximum amount of class II MHC molecules from lo6 neuraminidase-treated cells. Generally, 2 x 1O’to 4 x lo7 cells were used per experimental point, requiring 200-400 ~1 of gel. The appropriate amount of RCA,zo-agarose was washed in TBST, the supernatant removed, and the gel resuspended in the cleared cell lysate. Bound proteins were collected after agitation of the gel at +4OC for 2 hr, the agarose beads were transferred to a 1 ml pipette tip blocked with glass

WI 114

wool, and the supernatant was collected. The gel was washed with 1 vol of TBST buffer containing protease inhibitors, which was pooled with the first supernatant; this constituted the fraction from which “unbound” class II molecules were subsequently isolated. The RCA,,agarose was washed with an additional 3 bed vol of TBST buffer and galactose-terminated proteins eluted in 2.5 bed vol of TBST buffer containing an additional 0.15 M NaCl (i.e., final concentration, 0.3 M) and 0.1 M lactose and collected as the “bound” fraction. Class II MHC molecules were precipitated from each fraction as described above with DA6.231 MAb. Gel Electrophoresis SDS-gel electrophoresis was performed either by the method of Laemmli (1970) or by the method of Shagger and von Jagow (1987) using gels containing 16.5% total acrylamide with 3% bis-acrylamide. A cyanogen bromide digest of myoglobin (BDH) was run as a molecular weight standard. After fixation and destaining, these gels were equilibrated in 10% methanol (v/v), 10% acetic acid (v/v), 5% glycerol (wl w), and 0.5% polyacrylamide (w/w) to prevent cracking during the drying stage. Class II MHC a8 complexes displayed on ~0.6 mm thick 15% gels were excised from the wet gel using as a guide an autoradiogram exposed for 2 days; the gel piece was equilibrated for 30 min in Tris-Tricine sample buffer containing 0.5% SDS. Pilot experiments showed that only 20%-30% of the radioactivity was lost during this equilibration. The SDS buffer was removed, the gel slice was heated in a sealed tube for 5 min at 95OC, and then it was loaded directly onto ~1 .O mm thick 16.5% Tris-Tricine gels. Other Methods Lactoperoxidase-catalyzed iodination was performed as previously described (Watts, 1985). Surface labeling was also performed using sulfosuccinimidyi-3(4-hydroxyphenyl) propionate (Pierce Chemicals) as described by Thompson et al. (1987). Trichloroacetic acid precipitation of the culture supernatants was performed as previously described (Watts and Davidson, 1988). Acknowledgments We thank A. Sheppard and N. Fairweather for tetanus toxin, K. Guy for DA6.231 antibody, A. Sette and G. P. Corradin for peptides, and J. Kaufmann, P. Dellabona, S. Demotz, K. Karjalainen, and A. Living stone for helpful discussion and comments on the manuscript. This work was supported by the MRC and the Wellcome Trust. The Base1 Institute for Immunology was founded and is supported by Hoffmann La Roche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

January

23, 1991;

revised

July

and transport 85, 3975-3979.

Braciale, T. J., and Braciale, tural themes and functional Brodsky, processing

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