Acute myeloid leukaemia cells secrete a soluble factor that inhibits T and NK cell proliferation but not cytolytic function--implications for the adoptive immunotherapy of leukaemia

Abstract

Evidence of an immune mediated graft-versus-leukaemia effect has led to the belief that T and NK cell based adoptive immunotherapy can constitute effective treatment for relapsed leukaemias. However, work on solid tumours has shown this strategy may be hampered, by an immune escape mechanism in which tumour secreted immunosuppressive factors compromise T and NK cell function. Indeed, acute myeloid leukaemia (AML) cells secrete immunosuppressive factors that block the synthesis of Th1 type cytokines in T cells. We demonstrate here that this immunosuppression, mediated by both HL60 AML cell line and primary AML blasts, inhibits T and NK cell proliferation but not cytolytic activity. Supernatants from HL60 cell line and primary AML blasts inhibited T cell proliferation to mitogenic and alloantigen stimulation but had no effect on cytolytic function. Similarly, the proliferation of NK cells to IL-2 and IL-15 stimulation was inhibited whilst their cytolytic function, shown by lysis of AML blasts, K562 and Daudi cells remained unaffected. The failure of T and NK cells to proliferate was not due to effector cell apoptosis. Indeed, removal of lymphocytes from the immunosuppressive environment partially restored their capacity to respond to mitogenic stimulation. T cells exposed to immunosuppressive supernatants did not increase expression of mitotic inhibitory proteins that arrest cell division, thereby ruling this out as a mechanism of operation for this immunosuppression. T cell expansion requires antigen stimulation, usually provided in the form of AML blasts, therefore our data suggest that NK cells may be more practical for the immunotherapy of AML.

Fig. 1

HL60 supernatant inhibits T cell proliferation. (a) Proliferation, as determined by ³[H]Thymidine uptake, of PBMCs (2 × 10⁵ cells/well) stimulated with PHA (□) or alloantigen () in the presence or absence of supernatant from HL60, U937 and KG1a AML cell lines. Control supernatant used from PBMCs and HEL fibroblasts. Error bars represent s.d. from the means of a minimum of six experiments using different donors. *P ≤ 0·001. (b) Titration of HL60 supernatant shows a dose dependent inhibition in a PHA stimulated proliferation assay. Error bars represent s.d. from the means of three experiments using different donors.

Fig. 2

HL60 supernatant has no effect on T cell cytolytic function but inhibits proliferation. (a–c) Percent specific lysis of untransfected HLA-A,B,C negative 721.221 cells (□) and 721.221 cells transfected with HLA-A*0201 () or HLA-B*0702 (▪) by three alloreactive CTL lines, at an E : T ratio of 5 : 1, after culture in the absence (CM) or presence of HL60 supernatant (HL60-S) for 72 h. E : T ratio was corrected for number of live cells. Data representative of four experiments. (d–f) Proliferation, as measured by thymidine uptake of each of the alloreactive CTL lines after stimulation with alloantigen and IL-2 in the absence and presence of HL60 supernatant for 72 h. Results are represented as s.d. of triplicates and are representative of four experiments.

Fig. 3

HL60 supernatant has no effect on NK cell lytic function. (a) Lysis of K562 target cells by NK cells maintained in HL60 supernatant ( HL60-S) or complete medium (□ CM) over a 72 h time-course. Data representative of three experiments. Error bars represent s.d. from triplicates. (b) Lysis of Daudi target cells by IL-2 activated NK cells exposed to HL60-S () or kept in complete medium (□ CM).

Fig. 4

HL60 supernatant inhibits NK cell proliferation but not cytolytic function. (a) Effect of HL60 supernatant on the proliferation of NK cells in response to IL-2 and IL-15 stimulation. CM, complete medium; HL60-S, HL60 supernatant. (b) Ability of these NK cells to kill primary AML blasts. CM; □ HL60-S.

Fig. 5

Mechanism of immunosupression – effect of HL60 supernatant on the expression of mitotic inhibitory proteins. (a) ELISA of HL60 supernatant for IL-10 (□) and TGF-β (). (b) Effect of HL60 supernatant on the expression of mitotic inhibitory protein p27/Kip 1 by T cells kept in complete medium (CM) or HL60 supernatant (HL60-S) for 72 h. Protein molecular weight markers used included lysozyme (14 kD), carbonic anhydrase (30 kD) and ovalbumin (46 kD). (c) The corresponding effect of HL60-S on T cell proliferation as determined by thymidine uptake. □ unstimulated; PHA stimulated.

Fig. 6

Primary AML cells also secrete factors impairing lymphocyte proliferation but not cytotoxic function. (a) Effect of supernatant from primary AML blasts on the proliferation of T cells to PHA stimulation. Error bars represent s.d. from triplicates. unstimulated; ▪ PHA stimulated – no supernatant; □ PHA stimulated plus supernatant; *P ≤ 0·01; **P ≤ 0·002. (b) Effect of AML supernatants (AML-2, 4, 7 and 9) known to inhibit T cell proliferation on NK lysis of K562 cells.