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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.

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Adoptive transfer of CX3CR1-positive T cells to mice bearing RKO tumors over-expressing Fractalkine/CX3CL1. a Weight of RKO-Mock or RKO-CX3CL1 tumors after adoptive transfer of GFP-T cells or CX3CR1-T cells. (b, c) mRNA expression of CD3 and CX3CR1 (human specific primers) from RKO-Mock or RKO-CX3CL1 tumors receiving GFP-T cells or CX3CR1-T cells. d Quantification of Fractalkine in the blood serum of mice bearing RKO-Mock or RKO-CX3CL1 tumors. e Quantification of Fractalkine in the serum of mice bearing NCI-H630 tumors or in non-tumor-bearing mice
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Fig5: Adoptive transfer of CX3CR1-positive T cells to mice bearing RKO tumors over-expressing Fractalkine/CX3CL1. a Weight of RKO-Mock or RKO-CX3CL1 tumors after adoptive transfer of GFP-T cells or CX3CR1-T cells. (b, c) mRNA expression of CD3 and CX3CR1 (human specific primers) from RKO-Mock or RKO-CX3CL1 tumors receiving GFP-T cells or CX3CR1-T cells. d Quantification of Fractalkine in the blood serum of mice bearing RKO-Mock or RKO-CX3CL1 tumors. e Quantification of Fractalkine in the serum of mice bearing NCI-H630 tumors or in non-tumor-bearing mice

Mentions: We repeated the same type of experiment in mice bearing tumors formed by RKO-CX3CL1 or RKO-Mock cells. Surprisingly, after transfer of CX3CR1-T lymphocytes we did not observe any reduction in tumor weight (Fig. 5a), nor were the tumors more infiltrated by T cells, as evident from CD3 and CX3CR1 mRNA expression in isolated tumor infiltrating cells (Fig. 5b, c). We suspected that the chemokine Fractalkine could be possibly shed in the circulation by RKO-CX3CL1 cells, thus abrogating the chemokine gradient between tumor tissues and the systemic circulation. Serum levels of Fractalkine in mice bearing RKO-CX3CL1 tumors were in fact very high (700 ng/ml) (Fig. 5d) while less than 1 ng/ml was detected in the sera of mice bearing NCI-H630 tumors (Fig. 5e). Furthermore, the lymphocyte analysis from single cell suspension of lung tissues, after adoptive transfer regimen, showed significantly more CD3 lymphocytes entrapped in the lungs of mice bearing RKO-CX3CL1 tumors compared to RKO-Mock tumors : 70 % CD3+ vs 50 %, (Additional file 3: Figure S3A). Of note, no significant difference was observed in the lung infiltrate of NCI-H630 tumors (Additional file 3: Figure S3B).Fig. 5


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)

Adoptive transfer of CX3CR1-positive T cells to mice bearing RKO tumors over-expressing Fractalkine/CX3CL1. a Weight of RKO-Mock or RKO-CX3CL1 tumors after adoptive transfer of GFP-T cells or CX3CR1-T cells. (b, c) mRNA expression of CD3 and CX3CR1 (human specific primers) from RKO-Mock or RKO-CX3CL1 tumors receiving GFP-T cells or CX3CR1-T cells. d Quantification of Fractalkine in the blood serum of mice bearing RKO-Mock or RKO-CX3CL1 tumors. e Quantification of Fractalkine in the serum of mice bearing NCI-H630 tumors or in non-tumor-bearing mice
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Adoptive transfer of CX3CR1-positive T cells to mice bearing RKO tumors over-expressing Fractalkine/CX3CL1. a Weight of RKO-Mock or RKO-CX3CL1 tumors after adoptive transfer of GFP-T cells or CX3CR1-T cells. (b, c) mRNA expression of CD3 and CX3CR1 (human specific primers) from RKO-Mock or RKO-CX3CL1 tumors receiving GFP-T cells or CX3CR1-T cells. d Quantification of Fractalkine in the blood serum of mice bearing RKO-Mock or RKO-CX3CL1 tumors. e Quantification of Fractalkine in the serum of mice bearing NCI-H630 tumors or in non-tumor-bearing mice
Mentions: We repeated the same type of experiment in mice bearing tumors formed by RKO-CX3CL1 or RKO-Mock cells. Surprisingly, after transfer of CX3CR1-T lymphocytes we did not observe any reduction in tumor weight (Fig. 5a), nor were the tumors more infiltrated by T cells, as evident from CD3 and CX3CR1 mRNA expression in isolated tumor infiltrating cells (Fig. 5b, c). We suspected that the chemokine Fractalkine could be possibly shed in the circulation by RKO-CX3CL1 cells, thus abrogating the chemokine gradient between tumor tissues and the systemic circulation. Serum levels of Fractalkine in mice bearing RKO-CX3CL1 tumors were in fact very high (700 ng/ml) (Fig. 5d) while less than 1 ng/ml was detected in the sera of mice bearing NCI-H630 tumors (Fig. 5e). Furthermore, the lymphocyte analysis from single cell suspension of lung tissues, after adoptive transfer regimen, showed significantly more CD3 lymphocytes entrapped in the lungs of mice bearing RKO-CX3CL1 tumors compared to RKO-Mock tumors : 70 % CD3+ vs 50 %, (Additional file 3: Figure S3A). Of note, no significant difference was observed in the lung infiltrate of NCI-H630 tumors (Additional file 3: Figure S3B).Fig. 5

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