Reduction of immature myeloid cells by treatment with all-trans-retinoic acid (ATRA) improves the immunotherapeutic effect of the combination of local radiation with CTLA-4 blockade

Abstract

Accumulated evidence supports the contention that ionizing radiation therapy (RT) effects on tumor-host interactions extend beyond its ability to eliminate the cancer cells within a tumor. The ability of RT to generate inflammatory signals, to enhance the penetration of antigen-presenting cells (APC) and effector T-cells into solid tumors, to free tumor-derived antigens from the dying cancer cells, and to enhance the expression of some surface receptors on the cancer cells that survive, may all be important determinants of the effects of RT on tumor immunogenicity. We have recently shown that RT can enhance the immunogenicity of the poorly immunogenic, spontaneously metastasizing mouse mammary carcinoma 4T1 (Int J Radiat Oncol Biol Phys. 2003 Oct 1;57(2 Suppl):S259). Blockade of the CTLA4 receptor by the 9H10 antibody on T-cells lowers their activation threshold and has been shown to induce powerful anti-tumor immunity to partially immunogenic tumors. We find that a single dose (12 Gy) of RT can convert the poorly immunogenic 4T1 mammary carcinoma which is otherwise refractory to the treatment with CTLA-4 blocking antibodies into a responsive phenotype. Thus, treatment of 4T1 tumor-bearing mice with local RT followed by 9H10 administration induces a CD8 T cell-mediated anti-tumor immune response that inhibits the metastatic growth in the lungs and extends the survival of the mice. However, mice eventually succumb to their disease. 4T1 tumor growth is associated with dysregulation of APC maturation resulting in accumulation of immature (Gr1+CD11b+) myeloid cells (iMy) with immune suppressive function. The aim of the present study is to test whether depletion of iMy can result in a stronger and longer-lasting anti-tumor immune response following treatment with the combination of RT and CTLA-4 blockade. BALB/C mice were injected on day 0 with the syngeneic mammary carcinoma cells 4T1 in the flank at a single s.c. site considered as the primary tumor and treatment started when the growing tumors were at least 5mm in average diameter. All-trans-retinoic acid (ATRA) or placebo pellets (5 mg, 21-day release) were implanted s.c. on Day 7. Mice were randomly assigned to four groups: 1) non-specific hamster Ig (Ig)+ placebo; 2)Ig+ATRA; 3) RT+9H10 + placebo; 4) RT+9H10 + ATRA. RT was delivered to the s.c. tumor at a single dose of 12 Gy. The monoclonal hamster antibody against mouse CTLA4, 9H10, was given i.p. at 200 ug 1, 4 and 7 days after RT. Mice were followed for tumor growth and survival. Survival differences among treatment groups were analyzed using a Weibull model. Growth of 4T1 tumors was associated with progressive accumulation of Gr1+CD11b+ iMy cells in the spleen (up to 20–40% of total splenocytes). iMy cells also infiltrated the tumor, representing ∼5% of total cells. ATRA implantation on Day 7 dramatically reduced the percentage of iMy in the spleen of tumor-bearing mice compared to placebo and increased the percentage of dendritic cells, as measured on Day 20. Relative to placebo, administration of ATRA resulted in a statistically significant increase in survival in the presence of RT+9H10 (p = 0.0013) but had no significant effect in its absence. Furthermore, in these mice with more advanced tumors, relative to Ig control, the treatment with RT+9H10 significantly improved survival when in combination with ATRA (p = 0.0001) but had no significant effect on survival when administered with placebo. Tumor-induced immunosuppression is a great obstacle to the development of more effective immunotherapy treatments. These results show that, like other tumor vaccination strategies, the effectiveness of local RT combined with CTLA-4 blockade is limited by the accumulation of iMy cells, and their elimination by ATRA may be required to improve the therapeutic effect of this new treatment strategy