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Potential approach to immunotherapy of chronic lymphocytic leukemia (CLL): enhanced immunogenicity of CLL cells via infection with vectors encoding for multiple costimulatory molecules

Claudia Palena, Kenneth A. Foon, Dennis Panicali, Alicia Gómez Yafal, Jarasvech Chinsangaram, James W. Hodge, Jeffrey Schlom and Kwong Y. Tsang

Article Figures & Data

Figures

  • Figure 1.

    Functional analysis of CLL cells after infection with rV-TRICOM or PUVA-rV-TRICOM. (A-B) Mixed lymphocyte reaction using allogeneic T cells from healthy donors (1 × 105 per well) as effectors and various PBMC preparations (1 × 104 per well) as stimulators. Results are expressed as cpm ± SEM for triplicate wells.

  • Figure 2.

    Phenotypic analysis of CLL cells after infection with either MVA-WT or MVA-TRICOM. Flow cytometry analysis of PBMCs from patients 1 (A) and 14 (B) uninfected or 24 hours after infection with 5 MOI of MVA-WT or 5 MOI of MVA-TRICOM. After infection with MVA-TRICOM, 2 populations of CLL cells that expressed either low or high levels of each costimulatory molecule were observed; these were designated here as “low MFI” (L) and “high MFI” (H) populations, respectively.

  • Figure 3.

    Blocking of allogeneic T-cell proliferation in response to MVA-TRICOM–infected CLL cells. (A-B) Allogeneic T cells from healthy donors were stimulated in the presence of MVA-TRICOM–infected CLL cells (patients 15 and 16, respectively) that were preincubated in the presence of 5 μg/mL monoclonal Ab directed against B7-1, ICAM-1, or LFA-3, or combination of Abs (as indicated in the figure). Error bars represent the standard error of the mean (SEM) for triplicate determinations. (C) Percentage of inhibition of proliferation of allogeneic T cells in response to MVA-TRICOM–infected CLL cells in the presence of various Abs or combination of Abs, for patients 16 (○); 12 (•); 18 (□);5(▪); 15 (▵). The line represents the average for the 5 patients analyzed. Statistical comparison between groups was based on Student 2-tailed t test.

  • Figure 4.

    In vitro–expanded T cells were able to proliferate in response to uninfected autologous CLL cells. T cells expanded in vitro after 3 rounds (21 days) of stimulation with autologous MVA-TRICOM–infected CLL cells and exogenous IL-2 were incubated in the presence of autologous, uninfected CLL cells. Panels A and B correspond to the results with 2 different T-cell cultures, established from 2 different patients with CLL (patients 8 and 11, respectively). Abs (20 μg/mL) directed against MHC class I, class II; or an IgG control were added to the uninfected CLL cells for 1 hour before the addition of the T cells. Results are expressed as Stimulation index, calculated as cpm (T cells + CLL cells) / cpm (T cells alone).

  • Figure 5.

    Specific CTL-mediated lysis of autologous CLL cells. CD8+ isolated T cells that were in vitro expanded by 3 rounds of stimulation with autologous MVA-TRICOM–infected CLL cells and exogenous IL-2 were assessed for their ability to lyse autologous, uninfected 111In-labeled CLL cells. (A) Four different T-cell cultures established from 4 CLL patients (patients 6, 7, 8, and 9) were assayed. (B) Abs (20 μg/mL) directed against MHC class I or an IgG control were added to 111In-labeled target cells for 1 hour before the addition of the T cells (patient 8). Statistical comparison between groups was based on Student 2-tailed t test. Error bars represent the SEM for triplicate determinations.

Tables

  • Table 1.

    Characteristics of patients

    PatientSexAge, yTreatmentWBC count, × 109/LRai stageCD38Interphase cytogenetics*
    1 M 55 None 20 1 NA
    2 M 85 Chlorambucil 68 3/4 NA NA
    3 M 47 Multiple 40 1 Normal
    4 NA NA NA NA NA NA NA
    5 F 70 None 60 0 NA NA
    6 M 47 None 30 1 NA
    7 F 54 Chlorambucil§ 16 0 + +12 13q
    8 M 57 None 200 1 NA 13q
    9 M 50 None 37 1 NA NA
    10 M 75 Multiple 100 3/4 13q
    11 M 61 None 69 1 + 13q 11q
    12 F 79 Multiple 34 1 NA NA
    13 F 69 None 107 1 + Normal
    14 M 52 Fludarabine# 200 2 + Normal
    15 F 52 None 42 1 NA NA
    16 F 57 None 300 1 + +12 t(1;6)
    17 M 75 Multiple** 21 2 + 13q
    18 F 57 None 219 1 13q 6q
    19 F 56 None 72 1 + +12
    • WBC indicates white blood cell; NA, information not available.

    • * The only patients from which variable heavy chain (VH) gene mutational status was available were patients 8 and 10, both having VH unmutated

    • Rituximab, cyclophosphamide, vincristine, and prednisone (2000)

    • Patient 4 entered the study in condition of anonymity

    • § Treatment received in 2003 and 2004

    • Fludarabine/mitoxantrone (1999); cyclophosphamide, vincristine, and prednisone/rituximab (2003); alemtuzumab (2003)

    • Chlorambucil (1992–2000); rituximab, cyclophosphamide, vincristine, and prednisone (2002)

    • # Treatment received in 2001

    • ** Pentostatin/cyclophosphamide/rituximab (2003 and 2004)

  • Table 2.

    Flow cytometry analysis of PBMCs from patients 10 and 11 at 24 hours after infection with 10 MOI V-WT, PUVA-V-WT, rV-TRICOM, or PUVA-rV-TRICOM

    Patient, vectorB7-1, % (MFI)ICAM-1, % (MFI)LFA-3, % (MFI)
    Patient 10
       Uninfected 12.0 (22) 19.9 (31) 38.9 (30)
       V-WT 17.1 (33) 23.7 (28) 37.6 (22)
       PUVA-V-WT 23.5 (17) 17.4 (28) 41.4 (29)
       rV-TRICOM 39.9 (130) 54.9 (483) 49.6 (112)
       PUVA-rV-TRICOM 27.5 (116) 27.5 (125) 39.5 (39)
    Patient 11
       Uninfected 18.1 (31) 81.1 (62) 48.4 (32)
       V-WT 15.4 (34) 76.7 (37) 36.1 (20)
       PUVA-V-WT 13.6 (43) 75.7 (29) 45.0 (19)
       rV-TRICOM 52.8 (237) 96.3 (1055) 75.1 (168)
       PUVA-rV-TRICOM 38.3 (129) 89.1 (414) 51.9 (73)
    • Percentages are for cells that were positive for CD19 and each costimulatory molecule (B7-1, ICAM-1, and LFA-3). MFI values are for the expression of each costimulatory molecule.

  • Table 3.

    Phenotypic analysis of PBMCs from patients with CLL 24 hours after infection with MVA-TRICOM

    Patient, PBMCsB7-1, % (MFI)ICAM-1, % (MFI)LFA-3, % (MFI)
    1
       Uninfected 0.9 (74) 9.7 (25) 61.1 (28)
       MVA-TRICOM 61.7 (753) 76.9 (2310) 88.1 (1047)
    2
       Uninfected 1.0 (100) 29.2 (71) 21.6 (95)
       MVA-TRICOM 36.8 (1239) 67.2 (3430) 57.3 (1339)
    3
       Uninfected 17.7 (7) 83.2 (9) 81.2 (24)
       MVA-TRICOM 44.5 (106) 94.2 (134) 85.1 (67)
    4
       Uninfected 0.1 (31) 39.0 (137) 41.0 (118)
       MVA-TRICOM 10.5 (1384) 45.5 (2318) 44.4 (1053)
    5
       Uninfected 0.7 (192) 2.6 (118) 13.4 (25)
       MVA-TRICOM 69.7 (243) 67.2 (489) 66.6 (216)
    6
       Uninfected 10.7 (16) 71.3 (23) 96.3 (35)
       MVA-TRICOM 38.5 (383) 79.4 (906) 93.8 (351)
    7
       Uninfected 52.1 (29) 83.6 (35) 97.2 (99)
       MVA-TRICOM 72.4 (277) 92.2 (983) 97.2 (406)
    8
       Uninfected 0.1 (12) 97.5 (58) 66.0 (19)
       MVA-TRICOM 52.2 (348) 98.5 (1038) 89.2 (436)
    9
       Uninfected 1.9 (53) 18.3 (19) 14.4 (16)
       MVA-TRICOM 40.3 (200) 51.5 (506) 52.9 (216)
    10
       Uninfected 12.0 (22) 19.9 (31) 38.9 (30)
       MVA-TRICOM 64.3 (343) 95.8 (1474) 81.1 (539)
    11
       Uninfected 18.1 (31) 81.1 (62) 48.4 (20)
       MVA-TRICOM 78.9 (261) 97.1 (1321) 87.3 (468)
    12
       Uninfected 1.3 (141) 75.6 (53) 98.4 (62)
       MVA-TRICOM 92.6 (1052) 98.6 (1387) 99.9 (789)
    13
       Uninfected 0.4 (134) 78.7 (35) 88.4 (34)
       MVA-TRICOM 87.5 (477) 95.8 (952) 96.7 (597)
    14
       Uninfected 28.3 (135) 77.8 (213) 53.8 (96)
       MVA-TRICOM 84.9 (1113) 91.9 (1974) 95.9 (974)
    15
       Uninfected 7.1 (58) 37.2 (69) 77.0 (69)
       MVA-TRICOM 57.9 (880) 75.0 (2454) 88.6 (1026)
    16
       Uninfected 0.1 (116) 5.1 (86) 2.8 (98)
       MVA-TRICOM 39.3 (491) 45.5 (1049) 40.7 (500)
    17
       Uninfected 0.7 (24) 67.3 (26) 98.7 (54)
       MVA-TRICOM 82.4 (1378) 89.6 (1615) 92.7 (865)
    18
       Uninfected 0.7 (29) 22.1 (82) 90.5 (24)
       MVA-TRICOM 88.5 (601) 86.9 (631) 98.2 (330)
    19
       Uninfected 0.3 (346) 29.9 (47) 31.6 (35)
       MVA-TRICOM 82.5 (407) 68.4 (1062) 92.3 (491)
    • Percentages indicate positive cells for CD19 and B7-1, ICAM-1, or LFA-3; MFI values are for the expression of each costimulatory molecule. There were no changes in B7-1, ICAM-1, and LFA-3 expression in CLL cells from the above 19 patients when infected with wild-type MVA-WT vector.

  • Table 4.

    Flow cytometry analysis of PBMCs from patients 1 (A) and 14 (B) uninfected or 24 hours after infection with 5 MOI of MVA-WT or 5 MOI of MVA-TRICOM

    VectorB7-1, % (MFI)ICAM-1, % (MFI)LFA-3, % (MFI)
    Patient 1
       Uninfected 4.9 (23) 22.2 (18) 83.4 (25)
       MVA-WT 3.1 (16) 13.6 (16) 83.5 (25)
       MVA-TRICOM (combined) 68.9 (672) 82.0 (2131) 92.6 (987)
       MVA-TRICOM (low MFI) 36.5 (20) 62.0 (32) 88.6 (33)
       MVA-TRICOM (high MFI) 95.9 (920) 96.2 (3226) 96.1 (1949)
    Patient 14
       Uninfected 0.7 (2) 26.9 (63) 69.9 (36)
       MVA-WT 0.2 (2) 18.7 (50) 73.7 (27)
       MVA-TRICOM (combined) 81.8 (979) 93.1 (2476) 95.0 (1095)
       MVA-TRICOM (low MFI) 40.3 (43) 70.5 (50) 88.4 (45)
       MVA-TRICOM (high MFI) 98.8 (1534) 99.6 (3133) 99.2 (1766)
    • The percentage of double-positive cells (for CD19 and each costimulatory molecule), as well as MFI value for the expression of each costimulatory molecule (numbers in parentheses), are shown. Data on the combined populations were calculated as the percentage of double-positive cells in the entire PBMC population; data on the low MFI and high MFI populations were calculated after gating on each individual population of CLL cells.

  • Table 5.

    Allogeneic T-cell proliferation in the presence of MVA-TRICOM–infected CLL cells

    Patient, stimulator cellsAllogeneic proliferation stimulation index
    1
       Uninfected 1.0
       MVA-WT 1.3
       MVA-TRICOM 131.0
    2
       Uninfected 0.6
       MVA-WT 0.7
       MVA-TRICOM 121.8
    4
       Uninfected 0.6
       MVA-WT 0.8
       MVA-TRICOM 68.6
    16
       Uninfected 11.7
       MVA-WT 9.4
       MVA-TRICOM 34.4
    17
       Uninfected 2.9
       MVA-WT 2.9
       MVA-TRICOM 179.6
    18
       Uninfected 9.0
       MVA-WT 3.1
       MVA-TRICOM 35.8
    • CD3+ T cells from healthy donors were stimulated with uninfected, MVA-WT– or MVA-TRICOM–infected CLL cells, at a ratio of stimulator to effector cells equal to 1:10. Proliferation was measured as 3H-thymidine incorporation on day 5 of culture. Stimulation index was calculated as cpm (T cells + CLL cells)/cpm (T cells alone).

  • Table 6.

    Autologous T-cell proliferation in the presence of MVA-TRICOM–infected CLL cells

    Patient, stimulator cellsAutologous stimulation index
    2
       Uninfected 1.0
       MVA-WT 0.5
       MVA-TRICOM 34.8
    8
       Uninfected 1.3
       MVA-WT 1.6
       MVA-TRICOM 30.6
    16
       Uninfected 1.7
       MVA-WT 1.3
       MVA-TRICOM 27.2
    13
       Uninfected 1.1
       MVA-WT 1.0
       MVA-TRICOM 13.4
    14
       Uninfected 1.3
       MVA-WT 0.7
       MVA-TRICOM 5.3
    12
       Uninfected 1.1
       MVA-WT 1.3
       MVA-TRICOM 4.2
    19
       Uninfected 1.7
       MVA-WT 1.3
       MVA-TRICOM 2.3
    10
       Uninfected 0.7
       MVA-WT NA
       MVA-TRICOM 2.2
    18
       Uninfected 1.0
       MVA-WT 1.1
       MVA-TRICOM 1.9
    17
       Uninfected 1.1
       MVA-WT 1.3
       MVA-TRICOM 1.4
    • CD3+ T cells from patients with CLL were stimulated with uninfected, MVA-WT-, or MVA-TRICOM–infected CLL cells, at a ratio of stimulator to effector cells equal to 1:2.5. Proliferation was measured as 3H-thymidine incorporation on day 5 of culture. Stimulation index was calculated as cpm (T cells + CLL cells)/cpm (T cells alone).

      NA indicates not available.

  • Table 7.

    IFN-γ production by T cells from patients with CLL stimulated with autologous CLL cells

    IFN-γ (pg/mL)
    PatientProliferation in response to MVA-TRICOMUninfectedMVA-TRICOM
    8 High 0.0 748.2
    7 High 43.8 332.8
    11 High 37.4 220.8
    16 High 12.9 80.8
    18 Low 41.6 50.2
    14 Intermediate 0.0 0.0
    10 Low 0.0 0.0
    • CD3+ T cells from patients with CLL were stimulated (ratio of stimulator to effector cells, 1:2.5) with autologous CLL cells uninfected or infected with MVA-TRICOM. Supernatants were analyzed at 48 hours after stimulation.

      IFN-γ indicates interferon γ.