1. by Anja Freese, and Nicholas Zavazava
HLA-B7 β-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses
by Anja Freese, and Nicholas Zavazava
Blood
Volume 99(9):
May 1, 2002
©2002 by American Society of Hematology

2. Sequence alignment of the synthetic HLA-B7–derived peptides
Sequence alignment of the synthetic HLA-B7–derived peptides.Peptides covering residues 56 to 120 were synthesized using the FMOC technology.
Sequence alignment of the synthetic HLA-B7–derived peptides.Peptides covering residues 56 to 120 were synthesized using the FMOC technology.
Anja Freese, and Nicholas Zavazava Blood 2002;99:
©2002 by American Society of Hematology

3. Allopeptides were immunogenic
Allopeptides were immunogenic.(A) Irradiated autologous PBLs were pulsed with allopeptides and cocultured with nonirradiated autologous PBLs. After 4 days,3H-thymidine uptake was measured.
Allopeptides were immunogenic.(A) Irradiated autologous PBLs were pulsed with allopeptides and cocultured with nonirradiated autologous PBLs. After 4 days,3H-thymidine uptake was measured. P10 and P12 induced significant cell proliferation (P > .0001). Cells incubated with allogeneic stimulator cells (MLC) but no peptides showed, as expected, the highest proliferation. (B) To test whether APCs were required for peptide presentation, PBLs, purified T cells, APCs, and a mixture of reconstituted APCs and T cells were incubated with P10, and 3H-thymidine uptake was measured. Proliferation was only measured in cells containing APCs, suggesting the requirement of indirect presentation through the APCs. (C) Allopeptides augment allostimulation. Responder PBLs were stimulated in a 4-day mixed-day culture with irradiated allogeneic REH stimulator cells. Cell cultures were supplemented with various peptides, and [3H-thymidine uptake was measured. P10 and P12 induced elevated T-cell proliferation over the control cultures (Med) containing no peptides.
Anja Freese, and Nicholas Zavazava Blood 2002;99:
©2002 by American Society of Hematology

4. Allopeptides abrogate target cell lysis by anti–HLA-B7 CTLs
Allopeptides abrogate target cell lysis by anti–HLA-B7 CTLs.(A) Anti–HLA-B7 CTLs lysed HLA-B7 target cells but not non–HLA-B7 control cells.
Allopeptides abrogate target cell lysis by anti–HLA-B7 CTLs.(A) Anti–HLA-B7 CTLs lysed HLA-B7 target cells but not non–HLA-B7 control cells. (B) CTLs raised against stimulator cells efficiently lysed target cells, whereas CTLs raised against HLA-B7–derived synthetic peptides poorly lysed target cells.(C) P8, P10, and P11, but not P3 or P12, blocked cytolysis of target cells by anti–HLA-B7 CTL UWB7 in a concentration-dependent manner. (D) In contrast, P3 and P4—both HLA–A2-derived peptides—blocked target cell lysis by an anti–HLA-A2 CTL line, UWA2. P8, P10, and P11 (HLA–B7-derived peptides) did not modulate target cell lysis.
Anja Freese, and Nicholas Zavazava Blood 2002;99:
©2002 by American Society of Hematology

5. FITC-conjugated allopeptides enable visualization of alloreactive CTLs
FITC-conjugated allopeptides enable visualization of alloreactive CTLs.Anti–HLA-B7 CTLs HNB7.1 were incubated with FITC-conjugated P11 or control peptide P21 at 4°C, and fluorescence was measured by flow cytometry.
FITC-conjugated allopeptides enable visualization of alloreactive CTLs.Anti–HLA-B7 CTLs HNB7.1 were incubated with FITC-conjugated P11 or control peptide P21 at 4°C, and fluorescence was measured by flow cytometry. CTLs were successfully labeled by P11, but not by P21. To rule out peptide binding to the MHC alleles on the surfaces of CTLs rather than to the TCR, 2 CTL lines against A2 and B7, respectively, were raised from the same individual. P11 successfully labeled anti-B7 UWB7, whereas P21 labeled anti-A2 CTL only, indicating allospecific binding by the peptides.
Anja Freese, and Nicholas Zavazava Blood 2002;99:
©2002 by American Society of Hematology

6. CTL binding to allopeptides is temperature dependent
CTL binding to allopeptides is temperature dependent.Anti–HLA-B7 CTLs were labeled by P11 and were incubated at either 4°C or 37°C for 24 hours.
CTL binding to allopeptides is temperature dependent.Anti–HLA-B7 CTLs were labeled by P11 and were incubated at either 4°C or 37°C for 24 hours. At 4°C, the CTL binding to P11 was fairly stable for 12 hours but was much less stable at 37°C. Control peptide P21 remained low at both temperatures.
Anja Freese, and Nicholas Zavazava Blood 2002;99:
©2002 by American Society of Hematology