T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation

Abstract

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.

Figure 1

Chimerism in the peripheral blood MNC and PMN in patient EU 0–19 weeks after DLI. Alpha-fetoprotein (AFP) polymorphism was used to identify donor (D)-specific cells (black bars); factor IX to identify patient (P)-specific cells. Complete donor chimerism in the MNC was found 14 weeks after the DLI, whereas it was present in the PMN 2 weeks later.

Figure 2

Percentage specific lysis of patient leukemic cells in a ⁵¹Cr release assay (dotted bars) and percentage proliferation inhibition of CML progenitor cells from 10⁴ CML-MNC by three different CTL clones at effector/target ratios of 10:1, 3:1, and 1:1. The results are given as the mean of two experiments.

Figure 3

The proliferation of different numbers of unseparated CML-MNC, CD34⁺ CML progenitor cells, and CD34⁻ CML-MNC after 7 days of culture in progenitor cell culture medium. Proliferation was measured by [³H]thymidine incorporation (cpm) during 6 hr. Shown is the proliferation of the unseparated fraction (shaded bars), the proliferation of the CD34⁺ fraction (filled bars), and the CD34⁻ fraction (empty bars). The percentage of CD34⁺ cells was 1.9%, 6.2%, and 14.0% in patients HG, EU, and TO, respectively. In each tested concentration from each patient, the numbers of separated CD34⁺ and CD34⁻ cells tested were equivalent to the numbers of these cells in the unseparated cell fraction. All experiments were done in 6-fold.

Figure 4

MHC restricted recognition of target cells in the CML progenitor cell proliferation inhibition assay. A class II-restricted MHC antigen-specific CD4⁺ clone and a class I-restricted CD8⁺ clone were irradiated (10 Gy) and analyzed against CML-MNC at effector/target ratios of 4:1 and 1:1, respectively. Effector T cells or target CML-MNC were preincubated with control medium or antibodies against CD4, CD8, class I, or class II.

Figure 5

Frequencies of CML-PCILp and CTLp per ml of blood before or after DLI in patients EU, HG, and KK. Patient KK with a relapsed myeloid blast crisis entered complete remission 6 weeks after the first DLI. After 7 months a second relapse was treated unsuccessfully with IFNα and additional DLI. P indicates treatment with methylprednisolon. CR, complete remission.