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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

Analysis on tumor infiltrating human T cells after adoptive transfer to mice bearing NCI-H630 tumors. a mRNA expression of CD3, CD4, CD8 and CX3CR1 (human specific primers) from tumors of mice receiving eGFP-T cells (white bars) or CX3CR1-eGFP T cells (black bars), triplicates +/−SEM. b Immunohistochemical analysis of CD3 expression in paraffin embedded tumors after adoptive T cell transfer; c CD3 stain positive area quantified using image pro analysis software. d Weight of tumors after adoptive transfer of eGFP/CX3CR1-eGFP lymphocytes in mice (6–7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 for difference between eGFP and CX3CR1-eGFP expressing T lymphocytes (Student’s t test)
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Fig4: Analysis on tumor infiltrating human T cells after adoptive transfer to mice bearing NCI-H630 tumors. a mRNA expression of CD3, CD4, CD8 and CX3CR1 (human specific primers) from tumors of mice receiving eGFP-T cells (white bars) or CX3CR1-eGFP T cells (black bars), triplicates +/−SEM. b Immunohistochemical analysis of CD3 expression in paraffin embedded tumors after adoptive T cell transfer; c CD3 stain positive area quantified using image pro analysis software. d Weight of tumors after adoptive transfer of eGFP/CX3CR1-eGFP lymphocytes in mice (6–7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 for difference between eGFP and CX3CR1-eGFP expressing T lymphocytes (Student’s t test)

Mentions: The proportion of CD8+ and CD4+ subpopulation within CD3 positive TILs demonstrated that up to 85 % of cells expressed CD8 (Fig. 3c); furthermore, a greater proportion of CX3CR1+ T cells were present within the CD3-gated population (Fig. 3d). The FACS analysis obtained from four distinct mice showed significantly higher infiltration of CD3+ and CX3CR1+ lymphocytes in tumors of each mouse receiving CX3CR1-T lymphocytes, confirming their preferential tumor homing ability (Fig. 3e). The Real-time quantitative PCR also demonstrated significantly higher mRNA levels of T cell markers (CD3, CD4, CD8 and CX3CR1) in tumors of mice injected with CX3CR1-T cells (Fig. 4a). The presence of TIL was also investigated by immuno-histochemistry in tumor sections. We observed higher number of CD3 positive T cells in tumors of mice adoptively transferred with CX3CR1-T cells compared to mice receiving eGFP- T cells (Fig. 4b and c). Finally, the harvested NCI-H630 tumors were measured and we found significant reduction in tumor weight in mice injected with CX3CR1-T cells, indicating effective anti-tumor activity of receptor positive T lymphocytes (Fig. 4d).Fig. 4


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)

Analysis on tumor infiltrating human T cells after adoptive transfer to mice bearing NCI-H630 tumors. a mRNA expression of CD3, CD4, CD8 and CX3CR1 (human specific primers) from tumors of mice receiving eGFP-T cells (white bars) or CX3CR1-eGFP T cells (black bars), triplicates +/−SEM. b Immunohistochemical analysis of CD3 expression in paraffin embedded tumors after adoptive T cell transfer; c CD3 stain positive area quantified using image pro analysis software. d Weight of tumors after adoptive transfer of eGFP/CX3CR1-eGFP lymphocytes in mice (6–7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 for difference between eGFP and CX3CR1-eGFP expressing T lymphocytes (Student’s t test)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Analysis on tumor infiltrating human T cells after adoptive transfer to mice bearing NCI-H630 tumors. a mRNA expression of CD3, CD4, CD8 and CX3CR1 (human specific primers) from tumors of mice receiving eGFP-T cells (white bars) or CX3CR1-eGFP T cells (black bars), triplicates +/−SEM. b Immunohistochemical analysis of CD3 expression in paraffin embedded tumors after adoptive T cell transfer; c CD3 stain positive area quantified using image pro analysis software. d Weight of tumors after adoptive transfer of eGFP/CX3CR1-eGFP lymphocytes in mice (6–7 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 for difference between eGFP and CX3CR1-eGFP expressing T lymphocytes (Student’s t test)
Mentions: The proportion of CD8+ and CD4+ subpopulation within CD3 positive TILs demonstrated that up to 85 % of cells expressed CD8 (Fig. 3c); furthermore, a greater proportion of CX3CR1+ T cells were present within the CD3-gated population (Fig. 3d). The FACS analysis obtained from four distinct mice showed significantly higher infiltration of CD3+ and CX3CR1+ lymphocytes in tumors of each mouse receiving CX3CR1-T lymphocytes, confirming their preferential tumor homing ability (Fig. 3e). The Real-time quantitative PCR also demonstrated significantly higher mRNA levels of T cell markers (CD3, CD4, CD8 and CX3CR1) in tumors of mice injected with CX3CR1-T cells (Fig. 4a). The presence of TIL was also investigated by immuno-histochemistry in tumor sections. We observed higher number of CD3 positive T cells in tumors of mice adoptively transferred with CX3CR1-T cells compared to mice receiving eGFP- T cells (Fig. 4b and c). Finally, the harvested NCI-H630 tumors were measured and we found significant reduction in tumor weight in mice injected with CX3CR1-T cells, indicating effective anti-tumor activity of receptor positive T lymphocytes (Fig. 4d).Fig. 4

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