Introduction. In Chapter 5 it was shown that a high proportion of recently synthesised a/fî/Ii was isolated by anti-a^^. Therefore, vesicles derived from the antigen processing compartment were isolated, although they were not purified to homogeneity. Since these vesicles are intact (see chapter 4), they may contain molecules which perform important functions for antigen processing. In this chapter prel minary experiments are described to determine whether immuno-isolation could be
antigen processing.
ollowed by functional assays for
Firstly, using all the vesicular material isolated by anti-a^^, vesicles attached to ImAd were lysed by detergent, and the supernatant used to demonstrate li degradation. Secondly, formation of compact dimers after peptide loading was shown to be minimal for I-A^ and I-E^. In this regard, the indications were that A20 cells were not of the ideal MHC haplotype for further experiments.
li is degraded by immuno-isolated proteinases. Degradation of MHC II isolated by anti-a^yt was examined. A20 cells were either grown as usual (figure 42, lanes 1,2,7
and 8), or treated with leupeptin overnight to prevent li degradation in vivo (figure 42,
lanes 3,4,9 and 10). Both groups of cells were then pulsed with ^^S-amino-acids for 1 hour and chased for 2 hours, and membranes were isolated by anti-a^y^. The immuno-isolated material was then dissociated from ImAd, and incubated as follows: cells without leupeptin - incubated at either 4 “ C (lanes 1 and 7) or 37 “ C (lanes 2 and 8); cells with leupeptin - incubated at 37 " C either without (lanes 3 and 9) or with cathepsin B (lanes 4 and 10). Note that any cathepsin B in this immuno-isolated material would have been inhibited by leupeptin (in lanes 3 and 9), but that free leupeptin had been washed away and so exogenous cathepsin B would not be inhibited (in lanes 4 and 10). li-containing complexes were precipitated from all four samples by In-1 (figure 42, lanes 1 to 4). After
this clearing of a/6/Ii, remaining a/R in all four samples was precipitated by TIB 120
(figure 42, lanes 7 to 10). As a comparison, total cellular material was precipitated by In-1 (lane 5) and treated with cathepsin B (lane 6).
In this experiment, precipitation of total cellular MHC II by In-1 produced two major bands, corresponding to I-Ace and li (lane 5). The relative faintness of I-AB was unexplained. Cathepsin B treatment of MHC II precipitated by In-1 (lane 6) showed
to loss of a when the precipitation was via In-1. By comparison, precipitation via a led to loss of li only (see figure 26).
MHC II precipitated by In-1 from immuno-isolated material also showed two bands corresponding to I-Aa and li (lanes 1 to 4). The effect of incubation of MHC II together with eluted material from ImAd at 37 “ C can be seen by comparing lane 1 (4 “ C) and lane 2 (37 “C): degradation of li resulted from the incubation at 37 “C. The effect of resistance of MHC II to incubation at 37 " C per se^ and the susceptibility of immuno- isolated MHC II to cathepsin B can both be seen by from lanes 3 and 4. In lane 3 (37 “ C, cellular cathepsin B inhibited by leupeptin) the same amount of MHC II was recovered as from cells treated at 4 “ C (compare with lane 1). Therefore, raised temperature alone did not lead to loss of MHC II. In lane 4 (37 “ C, with exogenous cathepsin B) there was a reduction in MHC II (compare with lanes 3 and 1). Therefore, cathepsin B was capable of degrading isolated MHC II complexes as seen in lane 2.
a/i3 dimers precipitated sequentially after a/B/Ii from these samples showed characteristic a and 6 bands more prominently than li (lanes 7 to 10), as shown in chapter 4 (figure 24, lanes 3 and 4). No effect on the amount of a /6 precipitated was detected as a result of incubation at 37 “ C with cell-derived (co-isolated) cathepsin B (lane 8) compared to incubation at 4 “ C (lane 7). In addition, no effect on the amount of a /6 precipitated was detected as a result of incubation at 37 “C with exogenous cathepsin B (lane 10) compared to incubation at 37 ° C without active cellular cathepsin B (lane 9). This indicates that li was selectively lost due to incubation at 37 “C with co-isolated material (lane 2) or due to exogenous cathepsin B (lane 4), whereas a /6 was unaffected.
These results indicate that the isolated membranes contained a proteinase which degraded li. Therefore, an antigen processing activity was detected in material immuno- isolated by anti-a^y^. Further experiments could use intact vesicles to determine the extent of co-localisation of li and the proteinase. Such experiments might be optimised by using a chase period of 1 hour rather than 2 hours.
Compact dimers do not form after peptide-loading of I-A**. The formation of compact dimers follows peptide loading in some systems (Sadegh Nasseri and Germain, 1991), and is therefore a useful biochemical feature for monitoring antigen processing (Germain
and Hendrix, 1991; Davidson et al., 1991; Lanzavecchia et al, 1992; NeeQes and Ploegh,
compact dimers to a moderate extent (see figure 23), which is similar to the findings for I-A^ in splenocytes (Germain and Hendrix, 1991).
The formation of compact dimers in vitro was studied by incubating metabolically
labelled MHC II with a peptide restricted to I-A^: ova 323-339 (Shimonkevitz et aL, 1983;
Hunt et aL, 1992b). In two separate experiments MHC II attached to sepharose beads
was incubated at 37 “C firstly at pH 4.5, then at neutral pH for 15 minutes, before dividing the samples for SDS-PAGE (without 2-mercapto-ethanol) with and without boiling. The two experiments differed in the duration of exposure to peptide at low pH: either 10 minutes or two hours. The low pH should cause a large increase in both dissociation of previously bound peptides and binding of ova 323-339.
A20 cells labelled with a 1 hour pulse and a 1 hour chase provided MHC II which was incubated with peptide for 10 minutes. A faint, well-defined band was seen (figure 43, lane 1), which was partially sensitive to boiling (lane 2). The apparent molecular weight (61 kDa) was likely to be an under-estimate, since the band was displaced down the gel by a large amount of BSA. In this experiment, MHC II molecules not treated with peptide did not show the 61 kDa band (see figure 23, lanes 5 to 8).
MHC II in cells labelled with a 1 hour pulse and a 2 hour chase provided a mixture of
a/B /Ii and a/R. a/B/Ii was removed as in figure 24. The remaining a/B was incubated
with peptide for 2 hours. A band was seen (figure 43, lane 3), which was partially sensitive to boiling (lane 4) and had an apparent molecular weight of 67 kDa. In this experiment, MHC II molecules not treated with peptide showed the same SDS-resistant band, but to a lesser extent (figure 24, lane 4). The high molecular weight band in this experiment was more prominent than that seen in the previous experiment (figure 43, compare lanes 3 and 1). This difference might result less from the difference in duration of the incubations, and more from the higher proportion of MHC II complexes in lanes 3 and 4 which were a/B as opposed to a/B/Ii, which binds peptide with a much lower affinity (Roche and Cresswell 1990a).
It is likely that the band formed by peptide loading is identical to the band seen in boiled samples from metabolically labelled cells after 18 hours chase (figure 25, lane 6), since the bands have the same molecular weight and the same partial sensitivity to boiling. Therefore, I-A^ in A20 cells can form SDS-resistant a/B/peptide complexes, but with higher than the expected molecular weight for compact dimers (Sadegh Nasseri and Germain, 1991) and some resistance to boiling.
These results differ from a previous study which demonstrated compact I-A** dimers of
molecular weight «60 kDa which were completely sensitive to boiling (Dornmair et al,
1989). This difference might be explained by partial destabilisation of compact I-A^ by either 2-mercapto-ethanol in the tissue culture medium, or the manipulation of extracted MHC II in the absence of lipid micelles, together with a greater tendency of I-A^ to dissociate than other species of MHC II (Germain and Hendrix, 1991). This tendency is confirmed by observations which include: (1) the half-lives of peptide complexes with both I-A^ and I-E^ on live and fixed A20 cells and in vitro are quite short and can be
shortened by competing peptides (Pedrazzini et al, 1991; Poirier, 1992). (2) Compact
I-A^ dimers are more sensitive to temperature than I-E^ (Dommair et al, 1989).
In further experiments it would be interesting to attempt to form compact dimers using A20 cells which express a more suitable MHC haplotype. I-A*^ '^''®/I-A‘* A20 cells have been made in this laboratory (Poirier, 1992).
Future directions for the cell-free assay of antigen processing. The sub-cellular localisation of peptide loading to MHC II is not known. As discussed in chapter 5, it is likely that the vesicles in which peptide loading occur were isolated, although the preparation also contained vesicles from other intracellular MHC 11^^ compartments. Antigen processing could not be detected in this system by the appearance of compact dimers. Nevertheless antigen processing could be monitored by using labelled antigen and examining the recovery of intact or partially degraded antigen after immuno-isolation
with anti-ttj^ (Davidson et al, 1991; Marsh et al, 1992). Antigens used in such
experiments could be those for which the pattern of degradation has already been studied, for example ovalbumin or insulin. Alternatively antigens could be designed specifically, for example transferrin, HRP or anti-mouse Ig could be linked by sequences with known proteinase sensitivity to known epitopes. Such a system could also be used to study the enzymes which co-localise with MHC II by varying the susceptibility of the linker sequences.
Finally, the spectrum of molecules associated with MHC II in antigen processing could be studied by raising antisera to isolated vesicles and using standard techniques to clone molecules from an A20 expression library.
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8
9
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Figure 42: Degradation of li after immuno-isolation
A20 cells were either grown as usual (lanes 1,2,7 and 8), or treated with leupeptin
overnight to prevent li degradation in vivo (lanes 3,4,9 and 10). Both groups of cells were
pulsed with ^^S-amino-acids for 1 hour and chased for 2 hours, and membranes isolated by anti-ttj^. The immuno-isolated material was then dissociated from ImAd, and incubated as follows:
cells without leupeptin - incubated at either 4 “ C (lanes 1 and 7), or 37 “ C (lanes 2 and 8);
cells with leupeptin - incubated at 37 “ C either without (lanes 3 and 9), or with cathepsin B (lanes 4 and 10).
li-containing complexes were precipitated from all four samples by In-1 (lanes I to 4).
After this clearing of a/B/Ii, remaining a/R was precipitated by TIB 120 (lanes 7 to 10).
Also shown: total cellular li precipitated by In-1: untreated (lane 5), and treated with cathepsin B (lane 6).
M.W.
3
4
69— —^ IBP
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—
46
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30
—Figure 43: Incubation of I-A** with peptide to form compact dimers
Lanes 1 and 2: MHC II from metabolically labelled A20 cells (1 hour pulse, 1 hour chase) was precipitated by TIB 120, and incubated with peptide (ova 323-339) at pH 4.5 for 10 minutes. A faint, well-defined band (61 kDa, arrow) was seen which was partially sensitive to boiling. The band may be displaced down the gel by a large amount of BSA (69 kDa).
Lanes 3 and 4: MHC II from metabolically labelled A20 cells (1 hour pulse, 2 hours chase) was pre-cleared of a/ft/Ii with In-1, and a/8 precipitated by TIB 120. a/8 was incubated with peptide (ova 323-339) at pH 4.5 for 2 hours. A well-defined band (67 kDa, arrow) was seen which was partially sensitive to boiling. This band is more prominent than that seen after only 10 minutes incubation (compare lanes 3 and 1).