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. 2018 Apr 18;7(8):e1450715.
doi: 10.1080/2162402X.2018.1450715. eCollection 2018.

Chemokine receptor engineering of T cells with CXCR2 improves homing towards subcutaneous human melanomas in xenograft mouse model

Manja Idorn  1 , Signe Koggersbøl Skadborg  1 , Lauge Kellermann  2 , Hólmfrídur Rósa Halldórsdóttir  1 , Gitte Holmen Olofsson  1 , Özcan Met  1   3   4 , Per Thor Straten  1   4
Affiliations

Chemokine receptor engineering of T cells with CXCR2 improves homing towards subcutaneous human melanomas in xenograft mouse model

Manja Idorn et al. Oncoimmunology. .

Abstract

Adoptive cell therapy (ACT) using in vitro expanded tumor infiltrating T lymphocytes (TILs) from biopsy material represents a highly promising treatment of disseminated cancer. A crucial prerequisite for successful ACT is sufficient recruitment of transferred lymphocytes to the tumor site; however, despite infusion of billions of lymphocytes, T cell infiltration into the tumor post ACT is limited. By PCR and Luminex analyses we found that a majority of malignant melanoma (MM) cell lines expressed chemokines CXCL1/Groα, CXCL8/IL-8, CXCL12/SDF-1 and CCL2. Concerning expression of the corresponding receptors on T cells, only the IL-8 receptor, CXCR2, was not expressed on T cells. CXCR2 was therefore expressed in T cells by lentiviral transduction, and shown to lead to ligand specific transwell migration of engineered T cells, as well as increased migration towards MM conditioned medium. In vivo homing was assessed in a xenograft NOG mouse model. Mice with subcutaneous human melanoma were treated with MAGE-A3 specific T cells transduced with either CXCR2 or MOCK. Transducing T cells carrying the MAGE-A3a3a high affinity T cell receptor with CXCR2 increased tumor infiltration. Flow cytometry analysis 7 days after ACT showed a doubling in CD3+ T cells in tumor digest of mice receiving CXCR2 transduced T cells compared to MOCK treated mice, a finding confirmed by immunohistochemistry. In conclusion, our CXCR2 transduced T cells are functional in vitro and transduction with CXCR2 increases in vivo homing of T cells to tumor site.

Keywords: ACT; Adoptive cell therapy; CXCR2; Chemokine receptor; Genetic engineering; IL-8; Malignant melanoma; T cells.

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Figures

Figure 1.
Figure 1.
Chemokine profile of MM, and corresponding chemokine receptor expression on MM tumor infiltrating T cells. Initial analyses of mRNA expression in 20 melanoma cell lines of 3 select chemokines CCL2, CXCL8/IL-8 and CXCL12/SDF-1 by standard PCR (A), and subsequent luminex analyses of 11 select chemokines secreted into the supernatant of 22 MM cell lines after 48 h (B). Expression of corresponding chemokine receptors on CD4+ and CD8+ TIL either ex vivo, in young or REPd cultures analysed by 8-color flow cytometry (C). MM = Melanoma cell lines, n = 20 (PCR), 22 (Luminex); TIL = Tumor infiltrating lymphocytes, n = 10 Green = CCL2/CCR2; Blue = CXCL8/CXCR2; Red = CXCL12/CXCR4. Horizontal bars designate mean, error bars designate standard error of the mean.
Figure 2.
Figure 2.
In vitro functionality of T cells transduced with MAGE-A3a3a and CXCR2. Purity of transduced T cell cultures after sorting and in vitro expansion was assessed by flow cytometry (A+D). Transwell® migration of CXCR2 or Mock transduced T cells towards 0.5 ng/ml, 1 ng/ml, 5 ng/ml, 50 ng/ml and 100 ng/ml rh-IL8 (B), or MM supernatant (FM82) with or without 2 μg/ml neutralizing anti-IL-8 (C). Functional ability of tumor killing of MM cell line FM82 were assessed by 51Cr-release cytotoxicity assay, using decreasing effector:target ratios (from 90:1 to 0.1:1). (E) Light grey = untransduced control T cells, Dark grey = Mock transduced T cells; Blue = CXCR2 transduced T cells, DN = Double negative (untransduced). Effect of IL-8 signalling on tumor killing of FM82 was assessed by individual 51Cr-release of Mock (F) or CXCR2 (G) transduced T cells in combination with FM82 alone, FM82 + 5 ng/ml IL-8 or FM82 + 2 μg/ml anti-IL8. Effector phenotype of CXCR2 and Mock transduced T cells gating on EGFP_CXCR2 /vβ5.1 (MAGE-A3a3a TCR), CD3 /CD45RO, CD62 L /CCR7 (H).
Figure 3.
Figure 3.
In vivo homing of CXCR2 transduced T cells. CXCR2 expression of ACT infusion product was assessed by flow cytometry after sorting and expansion (A). Representative tumor growth curve (tumor surface mm2) measured by calipers. Red arrow indicated initiation of treatment (B). Frequencies of tumor infiltrating T cells were measured by flow cytometry in (C) or IHC (D) 7 days after ACT. Enumeration of tumor infiltration was performed by counting CD3+ 10 HPF at 600x magnification IHC (D-E). Circles in Fig. E highlight the representative IHC sections of PBS, CXCR2 or Mock T cell treated tumors depicted in D. Flow data (C) represents data from two independent experiments, each separately significant (P = 0.036 and P = 0.014), and together P = 0.0018. Light grey = PBS treated control mice, Dark grey = Mock transduced T cell treated, n = 13; Blue = CXCR2 transduced T cell treated, n = 11. Horizontal bars designate mean, and error bars the standard error of the mean. Unpaired t-test, *P < 0.05; **P < 0.01.
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Grants and funding

This work was supported by the Danish Cancer Society (R72-A4396-13-S2), Danish Council for Independent Research (DFF-1331-00095B).