The Antitumor Effect Of Irradiated Haploidentical Donor Lymphocytes Infusion

Donor lymphocyte infusions （DLI） have become an accepted method for enhancing the anti-tumor effect after bone marrow transplantation procedures. Recently, researchers found that single infusion of donor lymphocytes could induce graft versus tumor （GvT） effect. However, in clinical observations, singe infusion of donor lymphocytes without bone marrow transplant （BMT） could also induce lethal graft-versus-host disease （GvHD）. Ionizing radiation has been shown to limit the capacity of lymphocytes to clonal expansion while preserving the cytotoxicity of these lymphocytes against tumor cells thus better control or prevention of GvHD. Our previous study has shown that irradiated haploidentcal splenocytes and BM infusions could preserve anti-tumor effect and induce relatively weaker GvHD in parent C57BL/6→CB6F1 transplantation. The present study was designed to investigate the anti-tumor effect of 25Gy irradiated haploidentical DLI without a prior bone marrow transplantation （BMT） in BALB/c→CB6F1 infusion model systems, and to investigate its antitumor effect and possibly involved mechanism in CB6F1→CC3HF1 infusion model systems. The present study was composed of three parts: PartⅠEstablishment of subcutaneous tumor mouse model and the influence of different dose of irradiation on donor lymphocytes proliferation and cytotoxicity. PartⅡThe antitumor effect of 25Gy irradiated haploidentical lymphocyte infusions in Parents→F1 mouse model. PartⅢ:The antitumor effect and possibly involved mechanism of 5Gy irradiated haploidentical lymphocyte infusions in F1→F1 mouse model.Part I:Establishmen t of subcutaneous tumor mouse model and the influence of different dose of irradiation on donor lymphocytes proliferation and cytotoxicity.Aim The first study was designed to establish a hepa 1-6 tumor-bearing mice model suitable for irradiated haploidentical DLI. The second study was designed to investigate potential role of different dose of iron irradiation on lymphocytes proliferation and cytotoxicity. Methods Study one: Female CB6F1 mice were assigned to three groups （n=6 for each group）. CB6F1 mice were subcutaneously inoculated with 4×10⁶ hepa1-6 cells at the right flank. Tumor size was measured with calipers every 3 days. Tumor volume was calculated by the following formula: volume （mm³） = width²×length/2 （mm³）. The loss in total body weight was monitored every 3 days. The MHC phenotypes of hepa1-6 cells were assayed by the flow cytometry. When the tumor bearing mouse died, their ear skin, livers, small intestines and spleens were subjected to histopathological examination. Study two: Female BALB/c and CC3HF1 mice were sacrificed and spleens were removed aseptically. The splenocytes were prepared from the mice, and then the splenocytes were assigned to six groups, then irradiated 0Gy, 2.5Gy, 5Gy, 7.5Gy, 15Gy, 25Gy respectively. The proliferation of non-irradiated and irradiated splenocytes from CC3HF1 mouse was detected by MTT （3-（4,5）-dimethylthiahiazo （-z-y1）-3,5-diphenytetrazoliumrom） assay. Meanwhile, the cytotoxicity of non-irradiated and irradiated splenocytes from CC3HF1 mouse was analyzed by LDH assay.Results Study one: CB6F1 mice were subcutaneously inoculated with 4×10⁶ hepa1-6 cells at the right flank. The subcutaneous tumor can be detected on day 3 or 4. The three groups were devoid of any significant difference on the tumor growth and survival. We didn’t find the spontaneous remission in every group. In histopathological examination, the subcutaneous tumor was poorly differentiated carcinoma. The lung metastasis was found in tumor bearing mouse. Study two: After exposure to different dose of irradiation, the spleen cells were co-cultured with hepa 1-6 cells in the MLTC system. Irradiation splenocytes retained their cytotoxicity to hepa 1-6 cell and there was no significant difference between 5 Gy and higher dose of irradiation. These results indicated that irradiation had little influence on the cytotoxicity function of T cells and 5 Gy irradiation could significantly inhibit the proliferation of donor lymphocytes. Conclusions Part one: The hepa1-6 cell can grow in the CB6F1 mouse subcutaneous. Part two: Ionizing radiation could limit the capacity of lymphocytes to proliferate while preserving their cytotoxicity against tumor cells.PartⅡ:The antitumor effect of 25Gy irradiated haploidentical lymphocyte infusions in H-2^d→H-2^b/d mouse modelAim To determine whether 25Gy irradiated haploidentical DLI could induce GvT and inhibit GvHD in H-2^d→H-2_b/d mouse model and to investigate the role of donor derived T cell, CTX pretreatment and MHC in the antitumor effect.Methods Female CB6F1 mice were used as recipients, BALB/c, C3H mice were used as donors. Female CB6F1 mice were assigned to six groups （n=5 for each group）: PBS group （mice received a intravenous of PBS）, CTX group （mice received a intraperitoneal of cyclophosphamide）, TCD Sp/irr group （mice received a intravenous of irradiated BALB/c splenocytes in which T cells were deleted）, Sp/irr-BALB/c group （mice received a intravenous of irradiated BALB/c splenocytes）, Sp/irr-C3H group （mice received a intravenous of irradiated C3H splenocytes） and CTX+Sp/irr group （mice received a intravenous of cyclophosphamide combined with the irradiated BALB/c splenocytes） （n=5 for each group）. Tumor size was measured with calipers every 3 days. Tumor volume was calculated. GvHD was monitored by the loss in total body weight and confirmed by histology of the skin, small intestine, liver and spleen. Flow cytometric analysis of survival time of donor-derived lymphocytes and proliferation of tumor bearing mouse lymphocytes subset. Results: After 25Gy irradiated haploidentical DLI treatment to poorly immunogeneic hepa 1-6 tumor mouse model, the donor cells disappeared within 13 days. The infusion treatment could effectively inhibit the tumor growth and prolong the survival of recipients without inducing GvHD. This effect could be enhanced by the combined treatment with CTX, mismatched donor lymphocyte and impaired by deleting donor derived T cells.Conclusions 25 Gy irradiated haploidentical DLI could induce anti-tumor effect without GvHD in Parent→F1 infusion treatmen mouse model. Donor derived T cells play a crucial role in anti-tumor effect. The anti-tumor effect could be enhanced by pretreatment with CTX and using mismatched donor lymphocyte infusions.PartⅢ: Irradiated haploidentical donor lymphocyte infusions as an adoptive immunotherapy strategy to induce host-versus-tumor effects （F1→F1 infusion model systems）Aim To investigate the anti-tumor effect and the possibly involved mechanism of 5Gy irradiated haploidentical DLI in F1→F1 mouse model.Methods Female CB6F1 mice were used as recipients and CC3HF1 mice were used as donors. Female CB6F1 mice were assigned to five groups （n=5 for each group）: PBS group （mice received a intravenous of PBS）, CTX group （mice received a intraperitoneal of cyclophosphamide）, TCD Sp/irr group （mice received a intravenous of irradiated CC3HF1 splenocytes in which T cells were deleted）, Sp/irr- group （mice received a intravenous of irradiated CC3HF1 splenocytes）, and CTX+Sp/irr group （mice received a intravenous of cyclophosphamide combined with the irradiated CC3HF1 splenocytes） （n=5 for each group）. Tumor size was measured with calipers every 3 days. Tumor volume was calculated. GvHD was monitored by the loss in total body weight and confirmed by histology of the skin, small intestine, liver and spleen. At 3 days and 10 days after administration DLI, the tumor-bearing mice were killed at each time poin, and the cytotoxicity of splenocytes was analyzed in order to further evaluate the anti-tumor effect of irradiated DLI. Serum samples were collected from different groups of treated mice at each time point. IFN-γand IL-2 concentration were measured using specific ELISA kit for mouse IFN-γand IL-2. Flow cytometry was used to analyze the survival time of donor-derived splenocytes and the proliferation of host-derived lymphocytes subset.Results: After irradiated haploidentical DLI treatment to poorly immunogeneic hepa 1-6 tumor mouse model, the donor cells were rejected and disappeared within 4 days. Surprisingly, anti-tumor response was still observed. The infusion treatment could effectively inhibit the tumor growth and prolong the survival of recipients, and this effect could be enhanced by the combined treatment with CTX and impaired by deleting donor derived T cells. Moreover, the infusion treatment could increase the levels of type 1 cytokine including IFN-γand IL-2, and enhance the proliferation of lymphocytes subsets especially CD8⁺T and NK cells. Specifically, multiple infusions could enhance the anti-tumor effect without inducing GvHD.Conclusions As an adoptive therapy, 5Gy irradiated haploidentical DLI could induce host-versus-tumor effects absence of GvHD. Donor-derived T cells play a crucial role in this antitumor effect. CTX pretreatment could enhance the antitumor effect of infusion treatment absence of GvHD. Irradiated haploidentical DLI might be a promising and safe treatment for tumor.