Effective Adoptive Transfer Of Haploidentical Tumor-specific T Cells In B16-melanoma Bearing Mice

Adoptive immunotherapy is an appealing approach to cancer treatment,with the potential for more precise targeting and reduced toxicity. The mainclinical concern in using adoptively transferred self/tumor-specific T cells isthe lower possibility of graft-versus-host disease (GVHD). However,autologous tumor-specific T cells are often inconvenient to obtain, especiallyfrom patients with myeloma or with advanced disease. Adoptive transfer ofallogeneic tumor-specific T cells could provide an alternative to bridgingautologous T-cell therapy; however, its use could be hampered by GVHD. Thepersistence of transferred cells might be crucial for a favorable clinicaloutcome. Allogeneic cells with major MHC mismatches are rapidly rejectedby the host immune system. As haploidentical donor cells are more availablethan histocompatible allogeneic donor cells in clinical practice, we designedthe F1(H-d/k)S(H-2b/d) mice donor in our study to observe antitumor effectsand GVHD in a mouse model.Adoptive lymphocytes have been genetically modified in many ways toimprove activity and circumvent tumor evasion, including transfer oftransgenic T-cell receptors and chimeric antigen receptors to redirect T celland natural killer cell antigen specificity. Irradiation has been shown to reducethe capacity of lymphocytes while preserving the cytotoxicity of theselymphocytes against tumor cells, and can also limit GVHD response. Datafrom the research of Boni7showed that mice receiving a preparative regimenof myeloablating (9Gy) total body irradiation (TBI) experienced thesignificant regression of large, vascularized tumors, whereas mice receivingpreparative regimens of nonmyeloablating (5Gy) TBI experienced rapidrejection of tumor-specific allogeneic lymphocytes with no impact on tumorgrowth. In the present study, we pretreated B16-melanoma bearing C57BL/6mice with nonmyeloablating (7Gy) TBI without bone marrow transplantation(BMT), followed by adoptive transfer of haploidentical tumor-specific T cells.The results showed that use of tumor-specific haploidentical T cells can resultin significant antitumor effects, without severe GVHD. Here, we describe anovel adoptive therapy that could lead to a safe cancer treatment.Part1Influence of irradiation treatment on tumor regression andsurvival period of B-16bearing mice.Objective: Reportedly, allogeneic adoptive immunotherapy causedserious GVHD response, resulting in elevated mortality. Pre-treated recipientmice with chemotherapy or irradiation was reported to affect the tumormicroenvironment and result in immune inhibitory during tumorimmunotherapy. In this part, we sought to test whether irradiation had directeffects on tumor regression and survival period of B-16bearing mice.Methods: Models of tumor-bearing mice were established bysubcutaneous inoculation with melanoma cells (B16). C57BL/6mice bearingB16-melanoma tumors were irradiated with0,5,7Gy TBI, or7Gy TBI plussyngeneic bone marrow transplantation (BMT) with106unsorted bonemarrow cells the day after radiation (7Gy TBI+BMT). The dynamic changesof tumor size were observed and the growth curves were drawn.Results:1. The B16melanoma bearing mice were successfullyestablished7d after subcutaneous inoculation with B16melanoma cells inC57BL/6mice.2. Compared with the control group, there was no statisticaldifference of survival period in tumor-bearing mice from different groups.3. C57BL/6mice bearing B16-melanoma tumors were irradiated with0,5,7Gy TBI, or7Gy TBI pus BMT. Tumor areas were measured every3daysafter irradiation. There was no statistical difference in tumor growth betweenmice receiving5Gy TBI,7Gy TBI or7Gy TBI+BMT versus0Gy TBI.Conclusion: There was no statistical difference in tumor growth betweenmice receiving5Gy TBI,7Gy TBI or7Gy TBI+BMT versus0Gy TBI,which suggested that7Gy TBI did not influence tumor regression. Part2Effects of irradiation treatment on tumor microenvironment inB16-bearing mice.Objective: To observe the effects of irradiation treatment on tumormicroenvironment in B16-bearing mice.Methods: C57BL/6mice bearing B16-melanoma tumors were irradiatedwith7Gy TBI. Mouse blood white blood cells level was measured1,3,5,7,9,11,13d after irradiation. Quantitative detection of IL-10levels in mouseserum samples were measured using ELISA kit (Bender MedSystems GmbH,Vienna, Austria). In parallel experiments, total RNAs were isolated frommouse spleens, IL-10mRNA levels were measured by using real-time PCR.Foxp3mRNA levels in mouse spleens were measured by using real-time PCR.The percentage of CD4+CD25+Foxp3+T cells in mice spleens was analyzedby flow cytometry.Results:1. Peripheral blood white blood cell levels in B16bearingC57BL/6mice were significantly higher than those of normal mice. WhenB16bearing mice were treated with7Gy TBI, white blood cell levelsdecreased by86–96%from day1to day13after TBI.2. The IL-10proteinlevels in serum, and mRNA levels in spleen tissues of B16bearing mice usingELISA kit and real-time PCR, which reduced significantly after irradiationtreatment.3. Foxp3mRNA level in spleens of B16bearing mice weremeasured by real-time PCR. Foxp3mRNA levels decreased after7Gy TBIand reached their lowest level at day9after irradiation.4. Flow cytometrywas used to test the proportion of CD4+CD25+Foxp3+Tregs from spleencells of B16bearing mice. Compared with the non-irradiated group, Tregproportions significantly dropped from day1to day13after TBI, reachingtheir lowest level at day9, which was parallel with Foxp3mRNA levels.These results suggest that pre-treatment with7Gy TBI changes the tumormicroenvironment, causing an immune inhibitory effect in the B16bearingC57BL/6mice.Conclusion: These results suggest that pre-treatment with7Gy TBIchanges the tumor microenvironment, causing an immune inhibitory effect in the B16bearing C57BL/6mice.Part3Adoptive transfer of haploidentical DC-CTLs inhibitedB16-melanoma growth paralleled syngeneic DC-CTLs treatment.Objective: To investigate the effects of syngeneic or haploidenticalDC-CTLs treatment on B16tumor growth in mice and to explore whethertreatment with F1haploidentical DC-CTLs could induce GVHD-likereactions.Methods: C57BL/6mice bearing B16tumors established for10dayswere irradiated with7Gy TBI. Mice were injected with106Hoechst33342stained syngeneic DC-CTLs (Syn), or with106Hoechst33342stainedhaploidentical DC-CTLs (Hap) the day after irradiation. Hoechst33342stained syngeneic or haploidentical DC-CTLs in C57BL/6mice tumors orspleens were observed using fluorescence microscope. To investigate theB16-melanoma growth after syngeneic or haploidentical DC-CTLs transfer,C57BL/6mice bearing B16-melanoma were pre-irradiated with7Gy TBI.Mice were injected with PBS on both day0and day7(PBS [i.e., control]);with106syngeneic DC-CTLs on day0and PBS on day7(Syn1); with106haploidentical DC-CTL cells on day0and PBS on day7(Hap1); with106syngeneic DC-CTL cells on day0and day7(Syn2); or with106haploidentical DC-CTL cells on day0and day7(Hap2). Tumor areas weremeasured every3days after DC-CTLs treatment. The appearance of mice wasmonitored daily and assessed the degree of systemic GVHD by a scoringsystem. GVHD pathological manifestations were observed in the liver, lung,intestine and eye after DC-CTLs adoptive transfer.Results:1. Hoechst33342stained syngeneic or haploidentical DC-CTLsin C57BL/6mice tumors or spleens could be observed36h after adoptivetransfer. Fourteen days after adoptive transfer, nodosity alteration wasobserved in spleens of recipient mice.2. The tumor grew rapidly when micewere injected only with PBS. Both syngeneic and haploidentical DC-CTLstreatments significantly inhibited tumor growth from day9to day21. Whenmice were subjected to DC-CTLs twice (Syn2, Hap2), we observed more significant tumor regressions. However, there was no significant differencebetween syngeneic and haploidentical DC-CTLs transfer groups.3. A slightincrease of GVHD scores was seen in Syn2and Hap2mice on days15and21compared with PBS group, but there were no significant differences amongthe groups. On histopathological examination, no obvious lesions were seen ineyes, liver, lungs or intestines compared with PBS-treated mice.Conclusion: Either syngeneic or haploidentical DC-CTLs could persistin tumor or spleens in pre-irradiated B16bearing mice. HaploidenticalDC-CTLs treatment inhibits tumor growth, which paralleled syngeneicDC-CTLs treatment. The F1haploidentical DC-CTLs treatment gave nosignificant GVHD response.