Determinación de necesidades internas y externas sobre la información

(mean cpm) 20000- lL-2 release (mean cpm) 10000- Fi g u re 3.21 16hr cathepsin E digest at pH4.5 22504 Fraction number (1-40) 30000- 1 ohr cathepsin E digest at pH6.0 _{>50000 0}

l l i i i i i i i i i i i . i l , I I I , I , I I

Response o f IC5.1 to the fractionated peptide products o f a 16 hour cathepsin E digest of reduced HEL at pH4 5 vs pH6.0. Following separation on a C18 reverse-phase HPLC column, 5-10|j.l of each fraction was included in an IC5.1 hybridoma response assay, as detailed in methods and materials. IL-2 release was determined by standard CTLL assay. Results are expressed as the mean ^H-thymidine incorporation from triplicate cultures and are representitive of two separate experiments. Positive controls (red) are the response of IC5.1 to 1 OOpg/ml whole HEL. Negative controls (blue) show IL-2 release from IC5.1 in the presence of buffer, medium and APC alone .

o HEL HEL2-16 80000 60000 IL-2 release (mean cpm) 40000 20000 -o 100 3 10 30 0 0.3 1 Antigen concentration (pg/ml) Figure 3.22

R e s p o n s e of ED2 to HEL and synthetic H EL2-16 presen ted by Balb/c (I-A^) spleen cells. BD2 are re stricted murine T cell hybr ido m as , specific for the cry ptic de te rm in a nt , HEL2-16. ED2 and spleen cells were c o - cu lt ur ed in the p r e s e n c e of a range of ag co nc en tr ati on s for 16 hours and IL-2 release d e t e r m in e d by standa rd CTLL assay. Results are ex pr ess ed as the mean ^H- th y m id in e i n co r p or at io n from triplic ate cultures and are r e p re s en ta ti ve of four s ep ar at e exper im ent s.

15000- 10000- IL-2 release (mean cpm) Fraction number (1-40) 15000- 16hr cathepsin E 10000- IL-2 release (mean cpm) 5000-

l i i i i i i l l i l i i i l i l i i i i l i l l i l l l i . i l i l i i l l i l i

Response of ED2 T cell hybridomas to the fractionated peptide products of a partial and 16 hour cathepsin E digest of reduced HEL at ph4.5. S-lOpl of each isolated fraction were included in an ED2 hybridoma response assay, as detailed in methods and materials. IL-2 release was determined by standard CTLL assay. Results are expressed as the mean ^H-thymidine incorporation from triplicate cultures and are representative of three separate experiments. Positive controls (red) are the response of ED2 to lOOpg/ml whole HEL. Negative controls (blue) show IL-2 release from ED2 in the presence of buffer, medium and APC alone .

3.2.7 Aspartic proteinases can act as ‘a n ti-p rocessin g’ enzymes

The ability of cathepsins D and E to degrade a synthetic pe p ti d e spanning

the m aj or I-A^ HEL epitope (HEL46-61) is shown in figures 3.23 and 3.24.

Bot h enzymes are clearly able to destroy the epitope at pH4.5. However, d eg r ad at i o n by catheps in D is bl ock ed at pH5.0 or above, while cat hep si n E m ed ia te d pr oteolysis is only blocked at pH 5.5. As shown in figure 3.22, all d eg r ad at i o n was bl ock ed by p re -tr ea tm en t of peptid e with 50|iM pepstatin, c o n f i rm i n g that the observed enzym atic activity was not due to c o n ta m i n a ti n g non-aspartyl proteinases.

3.2.8 The expression of functional class II molecules in A20 and A20 derived B lym phoblastoid cells, A20UV and A20UVAV1.10, is distinct

As de t ai l ed in Me thods and Materials, two new cell lines have been derived from the A20 murine l ym pho bla sto id cell line in the laboratory.

D e m o n s tr a t e d in figure 3.26, wild type A20 express I-A^. A2 0U V, selected fro m u lt ra - v io le t light treated A20, have lost most of the ir class II

ex p r e s s io n (Poirier & Chain, 1993). A2 0 U V A V 1 .1 0 , whic h are I-A^

t ra n sf e c t e d A20UV, express I-A^. Vi ability of both the wil d type and

t r a n s f e c t e d class II molecules is co nf irm ed in Figure 3.27, which d em o n st r at es the re sponse of the I-A re stricted IC5.1 T cell h y b r id o m a and

the I-A^ re stricted AD71 T cell hy br idom a (detailed in table 2.1) to HEL p r e s e n t e d by each cell. A20 were clearly able to process and pr e se n t a range of HE L co nce nt ra ti ons to AD71, while A 20 U V A V 1 . 1 0 were shown to pr o ce ss and pr esent HEL to IC5.1. As expected, A20 UV were un able to p r e se n t HEL to either of the T cell hybridomas.

3.2.9 MHC II protects T cell epitopes from the ‘anti p ro c essin g ’ activity of cathepsin proteinases

As all three cathepsins tested were clearly able to act as ‘anti p r o c e s s i n g ’

en zym es in destroying the major I-A restr icted HEL epitope, HE L46 -6 1, the

effect of the presence of the I-A molec ule on the stability of this

d e t e r m in a n t was in vestigated Since pu rified I-A mole cul es were not

a va il ab le to us, the role of class II in rescuin g T cell d et er m in a nt s from d es tr u ct i v e proteolysis was studied in the conte xt of the intact cell. All cells were ex p o s ed to 1 mg/ml HEL for 16 hours and then lysed. The fractions r es ul ti ng from HPLC separation of each lysate were screene d for the

p r es en ce of an I-A restricted d et er m ina n t using the IC5.1 T cell hybr idom a,

and for an I-A^ restricted d ete rm in an t using the AD71 T cell hy br id om a

(d et aile d in table 2.1). Firstly, figures 3.29 and 3.30 d em o n st r at e that n ei th er of the determinants could be isol ate d from A20UV lysates. Figure 3.29 s hows that the I-A^ restricted de ter m ina nt, HEL46-61 coul d be detected by IC5.1 in the lysates obtained from I-A^ ex pr ess in g A 20 U V A V 1 . 1 0 , but

not in the lysates from I-A^ ex p r es s i n g A20. Similarly, figure 3.30

de m o ns tr at es that AD71 were able to detect the I-A^ re str ic te d HEL71-85

ep it op e from wild type A20, but not from the I-A^ ex pr es s in g