1. Patient HLA-DP–Specific CD4+ T Cells from HLA-DPB1–Mismatched Donor Lymphocyte Infusion Can Induce Graft-versus-Leukemia Reactivity in the Presence or Absence of Graft-versus-Host Disease 
Caroline E. Rutten, Simone A.P. van Luxemburg-Heijs, Constantijn J.M. Halkes, Cornelis A.M. van Bergen, Erik W.A. Marijt, Machteld Oudshoorn, Marieke Griffioen, J.H. Frederik Falkenburg 
Biology of Blood and Marrow Transplantation 
Volume 19, Issue 1, Pages (January 2013)
DOI: /j.bbmt
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Kinetics of different activation markers for detecting patient HLA-DP–specific CD4+ T cells directly ex vivo. PBMNCs from a patient (HLA-DPB1*02:01,03:01) who underwent allo-SCT for chronic B cell leukemia with an HLA-DPB1–mismatched donor (HLA-DPB1*04:02,05:01) were analyzed for the presence of HLA-DPB1*03:01–specific CD4+ T cells. Purified CD4+ T cells obtained during the clinical response to DLI were stimulated with HeLa-II cells transduced with donor- or patient-specific HLA-DPB1 molecules. (A) After 6-18 hours of incubation, intacellular IFN-γ (upper panel) or CD154 (lower panel) staining was determined by flow cytometry. (B) After hours of incubation, surface CD137 (upper panel) and CD154 (lower panel) expression was determined by flow cytometry. (C) Percentages of CD137-expressing CD4+ T cells after incubation with patient HLA-DPB1*03:01 (■), donor HLA-DPB1*04:02 (), or medium alone (□) was determined at different time points. Mean results ± SD of 2-4 individual experiments are shown.
Biology of Blood and Marrow Transplantation  , 40-48DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
Emergence of patient HLA-DP–specific CD4+ T cells after HLA-DPB1–mismatched allo-SCT and DLI. (A) Percentage of donor chimerism in bone marrow after allo-SCT. The arrow indicates DLI. Conversion to 100% donor chimerism was observed 3 months after DLI. (B) CD4+ T cells purified from donor PBMNCs, patient PBMNCs obtained after allo-SCT before DLI, and 6 weeks after DLI were stimulated with HeLa-II cells, HeLa-II cells transduced with donor HLA-DPB1*05:01 or shared HLA-DPB1*04:01, HeLa-II cells transduced with patient HLA-DPB1*01:01, or left unstimulated. Percentages of CD137-expressing CD4+ T cells after 44 hours of incubation are shown.
Biology of Blood and Marrow Transplantation  , 40-48DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
HLA-DP–specific CD137+/CD4+ T cells observed after HLA-DPB1–mismatched allo-SCT and DLI in 24 individuals. A total number of 24 patient–donor combinations were analyzed for the presence of patient HLA-DP–specific CD4+ T cells. In total, percentages of CD137+/CD4+ T cells in response to 39 donor or shared HLA-DPB1 molecules and 33 patient-specific HLA-DPB1 molecules are shown. For each patient–donor combination, CD4+ T cells purified from donor PBMNCs and patient PBMNCs obtained after allo-SCT before DLI and after DLI were stimulated with HeLa-II cells transduced with donor or shared HLA-DPB1 molecules (○) or HeLa-II cells transduced with patient-specific HLA-DPB1 molecules (●). Percentages of CD137+/CD4+ T cells are shown after 44 hours of coincubation with Hela-II cells. Results were corrected for background CD137 expression on unstimulated CD4+ T cells. For each patient–donor combination, representative results obtained after only 1 DLI are shown. HLA-DP–specific immune responses were defined as detection of >0.15% CD137+/CD4+ T cells after stimulation with Hela-II cells transduced with patient-specific HLA-DPB1 molecules based on <0.10% CD137+/CD4+ T cells in response to donor-specific or shared HLA-DPB1 molecules. The line represents the threshold of 0.15% for positive results. Results post-DLI represent the first DLI except for patients 14, 15, and 17. Results to the first DLI with a clinical response are shown for these patients.
Biology of Blood and Marrow Transplantation  , 40-48DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

5. Figure 4
HLA-DP–specific CD137+/CD4+ T cells in patients with and without clinical responses to DLI. Percentages of CD137+/CD4+ T cells in response to stimulation with HeLa-II cells transduced with patient HLA-DPB1 molecules obtained after allo-SCT and DLI are shown. Results were corrected for background CD137 expression on unstimulated CD4+ T cells. For 9 patients who were screened for CD4+ T cells specific for 2 different patient-specific HLA-DPB1 molecules, results for only 1 HLA-DPB1 molecule with the highest percentage of CD137+/CD4+ T cells are depicted. (A) Patient HLA-DP–specific CD4+ T cells were found in 13 of 18 patients with clinical responses to DLI and in 1 of 6 patients without clinical responses to DLI. (B) CD137+/CD4+ T cells were found in all 8 patients with GVHD (in presence or absence of GVL), and in 5 of 10 patients who developed beneficial clinical responses without GVHD (selective GVL reactivity). CD137+/CD4+ T cells were detected in only 1 patient without clinical response to DLI.
Biology of Blood and Marrow Transplantation  , 40-48DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions

6. Figure 5
HLA-DP–specific CD137+/CD4+ T cells in response to permissive and nonpermissive mismatches. Percentages of CD137+/CD4+ T cells in response to stimulation with HeLa-II cells transduced with patient HLA-DPB1 molecules obtained after allo-SCT and DLI are shown. Results were corrected for background CD137 expression on unstimulated CD4+ T cells. CD137+/CD4+ T cell responses to each HLA-DPB1 allele are depicted separately. The modified algorithm classifying HLA-DPB1 alleles in 4 categories was used [19]. Results of nonpermissive and permissive HLA-DPB1 mismatch combinations are shown. The horizontal line represents median values.
Biology of Blood and Marrow Transplantation  , 40-48DOI: ( /j.bbmt )
Copyright © 2013 American Society for Blood and Marrow Transplantation Terms and Conditions