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Article Figures & Data

Figures

  • Figure 1.

    Representative example demonstrating activation and expansion of γδ T cells during pamidronate/IL-2 treatment. Peripheral blood lymphocytes (PBLs) of patient 4B were stained with FITC- and PE-labeled antibodies and analyzed by flow cytometry before and after infusion of pamidronate and IL-2. Percentages of cells are given per quadrant. Body temperature (BT) was measured 2 times daily. Cytokine concentrations were determined in serum using ELISA systems. ND indicates not done.

  • Figure 2.

    Time course of responding patients. Response confirmed by (1) CT scan, (2) ultrasound, (3) PET scan, (4) blood tests, (5) lymph node, or (6) bone marrow biopsy. Embedded Image indicates treatment cycle; ⬢, relative tumor mass; SN, subcutaneous nodules; * lost to follow up.

  • Figure 3.

    Clinical response to treatment. CT scans of patients 8B and 9B before and after several cycles of pamidronate/IL-2 therapy show regression of skin metas tases and mediastinal lymph node (arrows), respectively.

Tables

  • Table 1.

    Patient characteristics and response to therapy

    γδ T-cell Proliferation
    Patient Age, y/sex Diagnosis/stage* Prior therapy (no. of cycles) Off therapy, mo IL-2 dose level (no. of cycles) Side effects§ In vitro In vivo Response
    Cohort A
    1A 83/M MM/III MP (15) 2 0.25-0.5 × 106 IU/m2 (2) PD
    2A 79/M CLL/IV CLB (10) 11 0.25-3 × 106 IU/m2 (6) F(1), T(3) + SD
    3A 67/F CLL/IV PM (10), CLB (5) 2 0.25-0.5 × 106 IU/m2 (2) I(1), F(1), T(2) PD
    4A 57/M IC/IV CLB/P (24), LR (30 Gy) 14 0.5 × 106 IU/m2 (1) T(2) PD
    5A 76/F MM/III MP (9) 10 0.5 × 106 IU/m2 (1) I(2), F(2), T(2) ND NE
    6A 63/F CLL/IV CLB/P (25), MCP (6), F (12), R (4) 5 0.5 × 106 IU/m2 (1) F(2), I(2) ND PD
    7A 68/M CLL/IV CLB/P (4), COP (6), CLB (12) 2 1-2 × 106 IU/m2 (2) I(2), F(2), T(3) ND PD
    8A 66/M MM/III MP (15), VID (6) 2 1 × 106 IU/m2 (1) ND PD
    9A 58/M MZL/III MCP (6), TBI/CY + PBSCT (1) 29 1 × 106 IU/m2 (1) F(2), T(2) ND PD
    10A 79/F MM/III CHOP (2), LR (50 Gy) 10 2-3 × 106 IU/m2 (2) F(1), T(2) ND PD
    Cohort B
    1B 73/M MM/III MP (2), VID (6) 3 0.25 × 106 IU/m2 (1) + PD
    2B 59/M MM/III MP (16), VID (6), HD-M + PBSCT (1), IFN 2 0.25 × 106 IU/m2 (2) T(2) + PD
    3B 52/M FCL/IV MCP (4), CHOP (2), Dexa-BEAM (2), IFN, LR (44 Gy), I131-R 4 0.25 × 106 IU/m2 (1) F(1), T(2) + + PD
    4B 43/F FCL/III TNI (49 Gy) 24 0.5-1 × 106 IU/m2 (4) F(2), T(2) + + + + + + SD
    5B 57/M MM/II VID (4) 2 0.5-2 × 106 IU/m2 (9) F(1), T(2) + + + + PR
    6B 36/F MM/II LR (30 Gy) 3 0.5 × 106 IU/m2 (1) F(1), S(1) + + + PD
    7B 46/M MZL/IV TNI (44 Gy) 29 1 × 106 IU/m2 (4) F(2), T(2) + + + SD
    8B 51/F FCL/IV COP (10), LR (30 Gy), MCP (4) 48 2 × 106 IU/m2 (4) F(1), T(2), S(2) + + + + PR
    9B 55/M FCL/III CHOP (4), Dexa-BEAM (2), TBI/CY + PBSCT (1), LR (30 Gy) 6 1-2 × 106 IU/m2 (8) F(1) + + PR
    • * MM indicates multiple myeloma; CLL, chronic lymphocytic leukemia; IC, immunocytoma; MZL, mantle zone lymphoma. Staging according to Durie and Salmon (MM), Rai (CLL), Ann-Arbor (IC; MZL).

    • MP indicates melphalan/prednisone; CLB, chlorambucil; PM, prednimustine; COP, cyclophosphamide/vincristine/prednisone; VID, vincristine/idarubicin/dexamethasone; MCP, mitoxantrone/chlorambucil/prednisone; CHOP, cyclophosphamide/vincristine/prednisone; F, fludarabine; R, rituximab; TBI/CY, total body irradiation/high-dose cyclophosphamide followed by peripheral blood stem cell transplantation (PBSCT); LR, local radiotherapy (dose); I131-R; radioimmunotherapy with iodine131-rituximab; Dexa-BEAM, dexamethasone/carmustine/etoposide/cytarabine/melphalan; HD-M, high-dose melphalan followed by PBSCT; TNI, total nodal irradiation (dose); and IFN, INF-α (maintenance therapy).

    • Months between last chemotherapy/radiotherapy and first pamidronate/IL-2 treatment.

    • § T indicates thrombophlebitis; F, fever; S, skin-erythema; I, infection; (2) WHO grade 2.

    • In vitro results represent percentages of control culture according to the following calculation: (γδ T-cell number in pamidronate/IL-2 culture)−(γδ T-cell number in medium/IL-2)/(γδ T-cell number in medium/IL-2) × 100. In vivo results represent percentage of increase according to the following calculation: (Vy9δ2 T-cell number on day 8 after pamidronate/IL-2 infusion)−(Vγ9δ2 T-cell number before treatment)/(Vγ9δ2 T-cell number before treatment) × 100. − Indicates <20%; +, 20% to 100%; + +, >100% to 200%; + + +, >200% increase of γδ T-cell number; ND, not done.

    • SD indicates stable disease; PR, partial remission; PD, progressive disease; NE, not evaluable.

    • No absolute counts are available; however, percentage of γδ T cells increased from 7% to 19.5%.

  • Table 2.

    In vitro proliferation of γδ T cells in response to pamidronate/IL-2

    Healthy donors (%) All patients (%) MM (%) NHL (%) B-CLL (%)
    Positive 14/16 (88) 20/41 (49) 12/26 (46) 8/10 (80) 1/5 (20)
    Negative 2/16 (12) 21/41 (51) 14/26 (54) 2/10 (20) 4/5 (80)
  • Table 3.

    Expression of activation antigens after pamidronate/IL-2 infusion of patients from cohort B

    αβ T cells NK cells γδ T cells
    Dose level, IL-2 IU/m2 Patient, n CD69, % mean (range) HLA-DR, % mean (range) CD69+ HLA-DR, n/patients (%) CD69, % mean (range) HLA-DR, % mean (range) CD69+ HLA-DR, n/patients (%) CD69, % mean (range) HLA-DR, % mean (range) CD69+ HLA-DR, n/patients (%)
    0.25 × 106 3 3 (−56-71) 10 (0-15) 0/3 (0) 24 (−17-43) 0 (0-123) 0/3 (0) 0 (−23-101) 163 (−7-375) 1/3 (33)
    0.5 × 106 3 33 (−23-95) 37 (27-86) 1/3 (33) 0 (−26-81) 0 (0-67) 0/3 (0) 53 (38-167) 400 (0-831) 2/3 (66)
    1 × 106 4 62 (3-153) 45 (0-122) 0/4 (0) 23 (18-27) 8 (−38-54) 0/2 (0) 183 (82-426) 322 (0-1253) 3/4 (75)
    2 × 106 3 65 (52-98) 60 (19-121) 2/3 (66) 81 (−37-103) 93 (79-291) 2/3 (66) 186 (62-230) 124 (122-571) 3/3 (100)
    • Results represent means of increase of the percentages of positive cells analyzed by double or triple staining (NK cells). CD69 was determined on day 2 or 3 and HLA-DR on day 7 after infusion. For analysis of patients expressing CD69 and HLA-DR, a threshold of 50% positive cells on day 8 was defined.

  • Table 4.

    Change of cell number of lymphocyte subpopulations after pamidronate/IL-2 infusion of patients from cohort B

    Increase γδ T cells, % mean (range)
    Dose level, IL-2 IU/m2 Patients, n Increase αβ T cells, % mean (range) Increase NK cells, % mean (range) Vδ1 (range) Vγ9δ2 (range)
    0.25 × 106 3 47 (−3-82) 44 (42-152) 64 (0-114) 0 (0)
    0.5 × 106 3 19 (11-23) 122 (19-136) 0 (−3-0) 128 (24-257)
    1 × 106 4 35 (7-70) 30 (0-98) 0 (0-36) 89 (−15-210)
    2 × 106 3 12 (−5-14) 37 (26-71) 0 (0-8) 57 (21-95)
    • Results represent means of increase in absolute cell numbers on day 7.

  • Table 5.

    In vivo proliferation of γδ T cells and response to treatment with pamidronate/IL-2 *

    In vivo γδ T-cell proliferation
    Objective response + Σ
    + 3 0 3
    2 13 15
    Σ 5 13 18
    • All analyzable patients (n = 18) according to Table 1 were included.

    • * P = .015, Fisher exact test.