In order to establish the optimal conditions required to induce a pp65- and CMV-specific
CTL response using synthetic peptides, several in vitro protocols were compared. PBMC
from a normal CMV seropositive and HLA-A*0201 positive individual designated as donor I (HLA A*0201, 3; B7, 60), were isolated, and used as "responder cells". Peptides AE42 and AE44 derived from pp65, which had previously shown affinity for the HLA-A2 molecule (Chapter 3), were used in these experiments. The influenza matrix peptide Flu-M 58-66 was also used as a positive control, since this peptide has previously been shown to be the dominant HLA-A2 restricted CTL epitope in the influenza matrix protein (Bednarek et al., 1991; Gotch e t a l , 1987).
In initial experiments, the induction of peptide-specific CTLs was performed using the antigen processing deficient cell line T2, pulsed with the synthetic peptides. The T2 cell line was chosen as it can be efficiently loaded with exogenously added peptides, and as shown in Chapter 3, the addition of CMV pp65 peptides AE42, AE44 or Flu-M 58-66 induces an increased level of HLA class I molecules on the surface of these cells. Therefore the possibility existed that the use of peptide-pulsed T2 cells as stimulator cells might propitiate the presentation of the appropriate peptide to CTLs.
Cultures were established using 2000 PBMCs per well, which were stimulated with irradiated, peptide-pulsed T2 cells, at a stimulator to responder cell ratio of 1:2.5. IL-2 was added to the cultures on day 3 and 6, before a secondary re-stimulation on day 10 with irradiated, peptide-pulsed T2 cells, IL-2 and irradiated autologous PBMC as feeder cells. This re-stimulation protocol was repeated weekly.
The cytotoxic potential of responder cells was assessed in a 4 h ^'Cr release assay on day 24. Preliminary experiments had suggested that T2 cells when used as stimulator cells, induced a very high background response and that the lysis attributed to this would
mask peptide-specific responses (data not shown). Thus in subsequent cytotoxicity
assays, to address the problem of high T2 backgrounds, a strategy using cold target lysis inhibition was employed. A ten-fold excess of non-^’Cr labelled (cold) T2 target cells, was added to each well containing ^*Cr labelled target cells, which were either non-pulsed T2 cells or T2 cells pulsed with the relevant peptide. This strategy allowed the lysis induced by potentially low frequency peptide-specific CTLs to be elucidated. Figure 5.1 shows that CTL lines generated against peptides Flu-M (58-66), and pp65 peptides AE42 and AE44 were able to demonstrate a peptide-specific response. Although in the case of CTLs stimulated with peptide AE44, the peptide-specific response was much lower than that observed with the other two peptides. The reactivity attributable to T2 cells alone was assessed by the use of a control in which only ^‘Cr labelled T2 cells were used as target cells.
a) Lines stimulated with T2 cells + pp65/AE42 peptide
O T 2 h o i A B 4 2 + T 2 c o l d 0 T 2 h o t + T 2 c o l d [ ] T 2 h o t
100
b) Lines stimulated with T2 cells + pp65/AE44 peptide
□ T 2 h o t ABU + T 2 c o l d Ü T 2 h o t + T 2 c o l d □ T 2 h o t 100
7 5
•a
c) Lines stimulated with T2 cells + Flu-M (58-66) peptide B T 2 h o t / F l u - M ( 5 8 - 6 6 ) + T 2 c o l d □ T 2 h o t 4 - T 2 c o l d [ ] T 2 h o t 1 0 0- 7 5 - c 5 0 - a . 2 5 -
j
I I I I r . - i c N r o ' ^ L n u 5 [ ' ~ o o c r \ o > H ( N > H r N n M ' L n ' x i r ' 0 0 c r i o < - ( C N i H ( N r o ' ^ L n ' ^ t ' ~ c o ( T i O . H ( N O O O O O O O O O . H r H r - I O O O O O O O O O . H r H i H O O O O O O O O O i - l i - H i H QQQQQDQQQQQQUCdWCdCdWWCdWWWWfafcfc,fc[i<[nCu&u[i<fc,6u,pMFigure 5.1 Initial reactivity o f polyclonal cultures generated using T2 cells pulsed with peptide.
PBM C from the HLA-A2 and CM V seropositive donor I were prim ed using irradiated T2 cells pulsed with the influenza matrix peptide Flu-M 58-66 (c) or the pp65 peptides AE42 (a) or AE44 (b). CTL activity was determined in a 4 h Chi'omium release assay by split well analysis, in which 150 pi was taken from each test well and split to be analysed against three different target cells: labelled T2 cells pulsed overnight with 50 p g /m l o f the appropriate peptide, or labelled T2 cells in the presence of a ten fold excess o f non-labelled T2 cells (cold) as com petitors, or labelled T2 cells alone. The figure show s the representative results from 36 out of 96 wells for each peptide.