Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug;24(8):751-60.
doi: 10.1089/hum.2013.051.

Cytosine Arabinoside Promotes Cytotoxic Effect of T Cells on Leukemia Cells Mediated by Bispecific Antibody

Affiliations
Free PMC article

Cytosine Arabinoside Promotes Cytotoxic Effect of T Cells on Leukemia Cells Mediated by Bispecific Antibody

Wei Li et al. Hum Gene Ther. .
Free PMC article

Abstract

Chemotherapeutic drugs can enhance an immune response of the host against the tumor in addition to killing cancer cells by direct cytotoxicity. Therefore, the combination of chemotherapy and immunotherapy is a promising approach for eliminating tumors, particularly in advanced stages. A strategic medication is to use a bispecific antibody format that is capable of recruiting polyclonal T cells around antibody-target-expressing tumor cells. Recently, we have constructed a bispecific antibody, anti-CD3×anti-CD19, in a diabody configuration. In this study, we measured B7 family members B7.1 (CD80) and B7.2 (CD86) expressed on a CD19(+) human leukemia cell line, Nalm-6, stimulated by cytosine arabinoside (Ara-C). We found that a low concentration of Ara-C could upregulate CD80 expressed on CD19(+) Nalm-6 cells. The cytotoxicity of T lymphocytes against Nalm-6 cells in vitro and in vivo mediated by the anti-CD3×anti-CD19 diabody with or without a low dose of Ara-C was compared. The combination of the anti-CD3×anti-CD19 diabody and Ara-C showed the greatest effectiveness in enhancing the cytotoxicity of T cells against the tumor cells in vitro and in vivo. Activated T cells expressed higher levels of CD25 and CD69 and released more interleukin 2. Both perforin/granzyme B system and Fas/FasL pathway were involved in the diabody-induced T-cell cytotoxicity. Moreover, the activated T cells could upregulate ICAM-3 expression on Nalm-6 cells, and inhibition of LFA-1-ICAM-3 interaction impaired cytotoxicity of T cells. It was noted that Ara-C could upregulate CD80 expressed on two of five specimens of acute B lymphoblastic leukemia patient-derived cells. Cytotoxicity of T cells against these two patient-derived cells was enhanced in the presence of the anti-CD3×anti-CD19 diabody. These findings indicate that treatment strategy using both cytotoxic lymphocyte-based immunotherapy and chemotherapy may have synergistic effects.

Figures

FIG. 1.
FIG. 1.
Growth inhibition of Nalm-6 cells by Ara-C in different concentrations and expression of CD80 and CD86 on Nalm-6 or B-ALL cells stimulated by Ara-C. (A) With the concentration increased, cytotoxicity of Ara-C increased. IC10 was 0.25 μM. (B) At the Ara-C concentration of 0.25 μM, CD80 increased along with prolonged time, but CD86 displayed no obvious change. (C) Stimulated by Ara-C (0.25 μM) for 72 hr, CD80 expression increased on two of five samples of B-ALL patient-derived cells. (D) CD86 expression displayed no obvious change on these five samples. **p<0.01. Ara-C, cytosine arabinoside; B-ALL, B lymphoblastic leukemia.
FIG. 2.
FIG. 2.
Cytotoxicity of human T cells, in different E/T ratios mediated by different concentrations of diabody in a nonradioactive cytotoxicity assay. (A) Cytotoxicity of T cells in the presence of Nalm-6 mediated by the diabody. (B) Cytotoxicity of T cells in the presence of Ara-C-stimulating Nalm-6 mediated by the diabody. The diabody was at different concentrations (10, 1.0, and 0.1 pM). E/T ratio ranged from 25:1 to 3:1. (C, D) Lysis of target cells by T cells mediated by PBS, diabody, anti-CD3scFv, or anti-CD19scFv alone; a mixture of anti-CD3scFv and anti-CD19scFv; and anti-CD3×anti-Pgp diabody. The concentration of a variety of antibodies was 10 pM. (C) Target cells were Nalm-6. (D) Target cells were Nalm-6 stimulated by Ara-C. (E) Lysis of B-ALL cells (sample 1) or B-ALL cells (sample 1) stimulated by Ara-C in the presence of diabody (10 pM) or not. E/T ratio ranged from 25:1 to 3:1. Data shown are the mean±SD of experiments performed in quadruplicate. E/T ratio, effector-to-target cell ratio; PBS, phosphate-buffered saline; SD, standard deviation.
FIG. 3.
FIG. 3.
Activation markers and cytokines release by activated T lymphocytes. The lysis of target cells was mediated by various antibodies at a concentration of 10 pM. Expression levels of T-cell activation markers CD25 (A) and CD69 (B) were significantly increased in case of the T cells incubated with target cells and the diabody. The T-cell activation markers increased at higher level when T cells were incubated with the Nalm-6 cells stimulated by Ara-C than Nalm-6 itself. Cytokine IL-2 significantly increased when T cells were incubated with target cells and the diabody. In addition, Nalm-6 cells stimulated by Ara-C could induce T cells to produce more IL-2 than Nalm-6 itself (C). No elevation of IL-4 compared with base line (D). *p<0.05 and **p<0.01 were compared with controls. T and T+diabody groups are controls.
FIG. 4.
FIG. 4.
Expression of perforin, granzyme B, and Fas ligand of activated T-cell subpopulation. There are greater percentage of perforin/granzyme B and FasL-expressing T cells after co-culturing tumors, T cells, and diabody when compared with the control. Nalm-6 cells preincubated with Ara-C stimulated more perforin (A)/granzyme B (B) and FasL (C) expressed by T cells than Nalm-6 cells alone. However, anti-CD3scFv or anti-CD19scF alone, combination of anti-CD3scFv and anti-CD19scFv, and anti-CD3/anti-Pgp have no effect. *p<0.05 and **p<0.01 were compared with controls. T and T+diabody groups are controls.
FIG. 5.
FIG. 5.
The functional significance of the involvement of ICAM-3. ICAM-3 expressed on Nalm-6 cells did not change after Nalm-6 cells were stimulated by Ara-C. ICAM-3 increased when Nalm-6 cells were stimulated by Ara-C co-cultured with T cells. When diabody was added, ICAM-3 increased significantly compared with the control (Nalm-6 group is a control) (A). The inhibition of the LFA-1–ICAM-3 interaction by anti-ICAM-3 mAbs and anti-LFA-1 mAbs reduced T-cell activation nearly to 45% on average. An isotype-matched control Ab did not decrease T-cell activation (B). *p<0.05 and **p<0.01 were compared with controls. mAbs, monoclonal antibodies.
FIG. 6.
FIG. 6.
Co-stimulation of molecular expression on Nalm-6 cells in vivo and antitumor effect of the anti-CD3×anti-CD19 diabody in xenotransplanted NOD/SCID nude mice. (A) CD80 expression was enhanced when mice were exposed to Ara-C of 1.0 mg/kg/day for 3 days but slightly enhanced for CD86. **p<0.01. (B) For the Nalm-6 xenotransplanted mice, a mixture of T cells and the diabody in different doses (5, 10, and 20 nM/mouse) were injected intravenously 24 hr later and once a week for 2 weeks. PBS, T cells alone, T cells combined with the mixture of anti-CD3scFv and anti-CD19scFv, and T cells combined with the anti-CD3×anti-Pgp diabody served as control groups.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

Feedback