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Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient.

Siddiqui I, Erreni M, van Brakel M, Debets R, Allavena P - J Immunother Cancer (2016)

Bottom Line: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses.Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells.Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Inflammation, Humanitas Clinical and Research Center, 20089 Rozzano, Milan Italy ; Ludwig Center for Cancer Research, Department of Oncology, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT

Background: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells.

Methods: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1.

Results: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus ifying the blood-tissue chemokine gradient.

Conclusions: This study demonstrates that ectopic expression of CX3CR1 enhanced the homing of adoptively transferred T cells towards CX3CL1-producing tumors, resulting in increased T cell infiltration in tumor tissues and decreased tumor growth. Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites.

No MeSH data available.


Related in: MedlinePlus

Transduction of the CX3CR1 receptor in human T cells and functional validation. a Population of CD3+ and of CD4+/CD8+ T cells post retroviral transduction of eGFP/CX3CR1 or -eGFP gene after 10 days of in vitro culture with hIL-2 medium. b Representative flow cytometry plot showing the transduction efficiency in eGFP or CX3CR1-eGFP in human T cells. Cells were gated on live cells followed by subsequent gating on CD3, CD4 and CD8 positive T cells. c mRNA expression of CX3CR1 in transduced CD3/IL-2 activated human T cells (Bars, Triplicates +/−SEM *P < 0.05, **P < 0.01 for difference between eGFP and CX3CR1-eGFP-T cells (Student’s t test). d Transwell migration assay of eGFP-T cells or CX3CR1-eGFP T cells in response to different concentrations of rhCX3CL1, ****P < 0.0001, Two-way ANOVA
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Fig2: Transduction of the CX3CR1 receptor in human T cells and functional validation. a Population of CD3+ and of CD4+/CD8+ T cells post retroviral transduction of eGFP/CX3CR1 or -eGFP gene after 10 days of in vitro culture with hIL-2 medium. b Representative flow cytometry plot showing the transduction efficiency in eGFP or CX3CR1-eGFP in human T cells. Cells were gated on live cells followed by subsequent gating on CD3, CD4 and CD8 positive T cells. c mRNA expression of CX3CR1 in transduced CD3/IL-2 activated human T cells (Bars, Triplicates +/−SEM *P < 0.05, **P < 0.01 for difference between eGFP and CX3CR1-eGFP-T cells (Student’s t test). d Transwell migration assay of eGFP-T cells or CX3CR1-eGFP T cells in response to different concentrations of rhCX3CL1, ****P < 0.0001, Two-way ANOVA

Mentions: The cDNA encoding CX3CR1-eGFP was cloned in the retroviral plasmid pMP71 and CD3-activated human T lymphocytes were transduced and further expanded in IL-2-containing medium. Three days post viral transduction, cells were >80 % CD3+ with a mixture of CD4 and CD8 lymphocytes (Additional file 2: Figure S2A); after 10 days, >90 % of cells were CD3+, indicating that the vast majority of IL-2-expanded cells were T lymphocytes, and 80 % were CD8+ (Fig. 2a).Fig. 2


Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient.

Siddiqui I, Erreni M, van Brakel M, Debets R, Allavena P - J Immunother Cancer (2016)

Transduction of the CX3CR1 receptor in human T cells and functional validation. a Population of CD3+ and of CD4+/CD8+ T cells post retroviral transduction of eGFP/CX3CR1 or -eGFP gene after 10 days of in vitro culture with hIL-2 medium. b Representative flow cytometry plot showing the transduction efficiency in eGFP or CX3CR1-eGFP in human T cells. Cells were gated on live cells followed by subsequent gating on CD3, CD4 and CD8 positive T cells. c mRNA expression of CX3CR1 in transduced CD3/IL-2 activated human T cells (Bars, Triplicates +/−SEM *P < 0.05, **P < 0.01 for difference between eGFP and CX3CR1-eGFP-T cells (Student’s t test). d Transwell migration assay of eGFP-T cells or CX3CR1-eGFP T cells in response to different concentrations of rhCX3CL1, ****P < 0.0001, Two-way ANOVA
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836203&req=5

Fig2: Transduction of the CX3CR1 receptor in human T cells and functional validation. a Population of CD3+ and of CD4+/CD8+ T cells post retroviral transduction of eGFP/CX3CR1 or -eGFP gene after 10 days of in vitro culture with hIL-2 medium. b Representative flow cytometry plot showing the transduction efficiency in eGFP or CX3CR1-eGFP in human T cells. Cells were gated on live cells followed by subsequent gating on CD3, CD4 and CD8 positive T cells. c mRNA expression of CX3CR1 in transduced CD3/IL-2 activated human T cells (Bars, Triplicates +/−SEM *P < 0.05, **P < 0.01 for difference between eGFP and CX3CR1-eGFP-T cells (Student’s t test). d Transwell migration assay of eGFP-T cells or CX3CR1-eGFP T cells in response to different concentrations of rhCX3CL1, ****P < 0.0001, Two-way ANOVA
Mentions: The cDNA encoding CX3CR1-eGFP was cloned in the retroviral plasmid pMP71 and CD3-activated human T lymphocytes were transduced and further expanded in IL-2-containing medium. Three days post viral transduction, cells were >80 % CD3+ with a mixture of CD4 and CD8 lymphocytes (Additional file 2: Figure S2A); after 10 days, >90 % of cells were CD3+, indicating that the vast majority of IL-2-expanded cells were T lymphocytes, and 80 % were CD8+ (Fig. 2a).Fig. 2

Bottom Line: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses.Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells.Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Inflammation, Humanitas Clinical and Research Center, 20089 Rozzano, Milan Italy ; Ludwig Center for Cancer Research, Department of Oncology, University of Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT

Background: Adoptive T-cell based immunotherapies constitute a promising approach to treat cancer, however, a major problem is to obtain effective and long-lasting anti-tumor responses. Lack of response may be due to insufficient trafficking of specific T cells to tumors. A key requirement for efficient migration of cytotoxic T cells is that they express chemokine receptors that match the chemokines produced by tumor or tumor-associated cells.

Methods: In this study, we investigated whether the in vivo tumor trafficking of activated T cells could be enhanced by the expression of the chemokine receptor CX3CR1. Two human colorectal cancer cell lines were used to set up a xenograft tumor model in immunodeficient mice; the NCI-H630, constitutively expressing the chemokine ligand CX3CL1 (Fractalkine), and the RKO cell line, transduced to express CX3CL1.

Results: Human primary T cells were transduced with the receptor CX3CR1-eGFP. Upon in vivo adoptive transfer of genetically modified CX3CR1-T cells in mice bearing NCI-H630 tumors, enhanced lymphocyte migration and tumor trafficking were observed, compared to mice receiving Mock-T cells, indicating improved homing ability towards ligand-expressing tumor cells. Furthermore, significant inhibition of tumor growth was found in mice receiving modified CX3CR1-T cells. In contrast, tumors formed by RKO cells transduced with the ligand (RKO-CX3CL1) were not affected, nor more infiltrated upon transfer of CX3CR1-T lymphocytes, likely because high levels of the chemokine were shed by tumor cells in the systemic circulation, thus ifying the blood-tissue chemokine gradient.

Conclusions: This study demonstrates that ectopic expression of CX3CR1 enhanced the homing of adoptively transferred T cells towards CX3CL1-producing tumors, resulting in increased T cell infiltration in tumor tissues and decreased tumor growth. Our results also establish that a correct chemokine gradient between the systemic circulation and the tumor is an essential requirement in adoptive T-cell based immunotherapy to efficiently recruit T cell effectors at the correct sites.

No MeSH data available.


Related in: MedlinePlus