Regulation of Tumor Growth by Leukocyte-Specific Protein 1 in T Cells
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Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001180 on 5 October 2020. Downloaded from Regulation of tumor growth by leukocyte-specificprotein1inTcells Riri Kwon,1,2 Bong- Ki Hong,1 Kang- Gu Lee,1,2 Eunbyeol Choi,1,2 Laurent Sabbagh,3 1,4 1 1,4 Chul- Soo Cho, Naeun Lee, Wan- Uk Kim To cite: Kwon R, Hong B- K, ABSTRACT determines tumor progression and the effi- Lee K- G, et al. Regulation of Background Clinical efficacy of T cell- based cancer cacy of antitumor immunotherapy, including tumor growth by leukocyte- immunotherapy is limited by the lack of T cell infiltration specific protein 1 inT cells. antibody (Ab)- based immunotherapy against in the tumor mass, especially in solid tumors. Our group 1–3 Journal for ImmunoTherapy programmed cell death protein 1 (PD-1). demonstrated previously that leukocyte- specific protein of Cancer 2020;8:e001180. Several studies have suggested that a high doi:10.1136/jitc-2020-001180 1 (LSP1), an intracellular signal regulator, negatively regulates T cell infiltration in inflamed tissues. density of T cells positively correlates with Methods To determine the immuno- regulatory effects favorable prognosis and survival in patients ► Additional material is published online only. To view, of LSP1 in T cells on tumor progression, we investigated with various cancers, including colorectal, please visit the journal online the growth of B16 melanoma in Lsp1 knockout (KO) mice non-small cell lung, hepatocellular, pancreatic (http:// dx. doi. org/ 10. 1136/ jitc- and T cell- specific Lsp1 transgenic (Tg) mice. The immune and gastric cancers and melanoma.1 2 There- 2020- 001180). cell subpopulation infiltrated into the tumor mass as well fore, adoptive cell transfer using antigen- as the expression of interferon-gamma (IFN-γ) and tumor activated T cells, particularly chimeric antigen Accepted 07 September 2020 necrosis factor- alpha (TNF-α) in T cells was assessed by flow cytometry and/or immunohistochemistry. Chemotactic receptor (CAR)- T cells, has emerged as one migration was assayed with Lsp1 KO and Lsp1 Tg T of the promising strategies to improve the 4 cells. Adoptive transfer of Lsp1 KO or Lsp1 Tg T cells was efficacy of anticancer therapy. For example, performed in B16 melanoma-challenged Rag1 KO mice. CD19- targeted CAR-T cell therapy has shown Results Lsp1 KO mice showed decreased growth of remarkably high rates of remission in patients B16 melanoma and increased infiltration of T cells in the with hematological malignancies, including tumor mass, which were completely reversed in T cell- + relapsed or refractory B- cell acute lympho- specificLsp1 Tg mice. Lsp1 KO CD8 T cells also exhibited 4 5 elevated migratory capacity in response to CXCL9 and blastic leukemia and lymphoma. CXCL10, whereas Lsp1 Tg CD8+ T cells did the opposite. Despite its success in hematological malig- LSP1 expression was increased in tumor- infiltrating T nancies, CAR- T cell therapy is not always effi- http://jitc.bmj.com/ cells and could be induced by T cell receptor activation. cacious and has shown rather disappointing © Author(s) (or their Intriguingly, gene expression profiling ofLsp1 KO T cells employer(s)) 2020. Re- use results in some patients with solid tumors. One permitted under CC BY. suggested enhanced cytotoxicity. Indeed, expression of + of the major hurdles of T cell- based cancer Published by BMJ. IFN-γ and TNF-α was increased in tumor- infiltrating CD4 + immunotherapies is insufficient trafficking 1 and CD8 T cells of Lsp1 KO mice, while it was markedly Center for Integrative of T cells into tumor masses.4 6 Despite the Rheumatoid Transcriptomics reduced in those of Lsp1 Tg mice. Adoptive transfer of infusion of large amounts of T cells after ex on September 25, 2021 by guest. Protected copyright. and Dynamics, The Catholic Lsp1 KO T cells to Rag1 KO mice was more effective in University of Korea, Seoul, suppressing melanoma growth than transfer of Lsp1 Tg vivo expansion, only a small portion of trans- Republic of Korea T cells. Of note, when treated with antiprogrammed cell ferred T cells reaches inside the tumor tissues 2 Department of Biomedicine & death protein 1 (PD-1) antibody, inhibition of melanoma in clinical and preclinical studies.7 Although Health Sciences, The Catholic growth was more pronounced in Lsp1 KO mice than in University of Korea, Seoul, it remains unclear why trafficking, infiltration Lsp1- sufficient mice, suggesting that Lsp1 depletion and penetration of T cells are insufficient, it Republic of Korea additively increases the antitumor effects of anti- PD-1 3Department of Microbiology, antibody. may be primarily because solid tumors shape Infectiology, and Immunology, more fibrotic and less invasive environments University of Montreal, Montreal, Conclusions LSP1 in T cells regulates the growth of B16 melanoma in mice, possibly by affecting migration and through the activation of tumor- associated Quebec, Canada 8 4Division of Rheumatology, infiltration of T cells into the tumor and by modulating fibroblasts, ultimately constructing immu- + Department of Internal Medicine, production of antitumor effector cytokines by CD8 T cells. nosuppressive tumor microenvironments The Catholic University of Korea, These findings provide evidence that LSP1 can be a target (TME). Thus, to maximize the efficacy of T Seoul, Republic of Korea to improve the efficacy of T cell-based immunotherapy. cell- based immunotherapy for solid tumors, Correspondence to it is essential to develop innovative ways for Professor Wan- Uk Kim; BACKGROUND the successful delivery of immunocompetent wan725@ catholic. ac. kr Immune contexture, which consists of the T cells inside the tumor mass by destroying Dr Naeun Lee; density, composition and functional status or detouring fibrotic and immunosuppressive nelee2015@ catholic. ac. kr of tumor- infiltrating leukocytes (TILs), TME.8 9 Kwon R, et al. J Immunother Cancer 2020;8:e001180. doi:10.1136/jitc-2020-001180 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001180 on 5 October 2020. Downloaded from Leukocyte- specific protein 1 (LSP1) is an intracellular then PCR analysis of the Lsp1 transgene was performed F- actin- binding protein that is mainly expressed in hema- using the following primer sequences: forward, 5’-GGAC topoietic cells, such as T and B lymphocytes, neutrophils TCCA CCAG TCTC ACTTCAG-3’ and reverse, 5’- CAGT and macrophages.10 Previous studies have reported a TCAG AGGA CTTC AGGCTGAT-3’. G protein signaling negative regulatory role of LSP1 in leukocyte recruitment 7 gene (Rgs7) was used as an internal control with the to inflamed sites.10–12 After peritoneal injection of thiogly- primers 5’- CAAC CACT TACA AGAG ACCCGTA-3’ and 5’- colate or intra- articular injection of zymosan, infiltration GAGC CCTT AGAA ATAA CGTTCACC-3’. of macrophages and neutrophils was found to be higher For the adoptive transfer experiments using T cells, in inflamed tissues of Lsp1 knockout (KO) mice than in Rag1 KO mice were obtained from Jackson Labora- those of wild-type (WT) mice.11 12 Recently, our group also tory (Bar Harbor, Maine, USA). All strains were in the demonstrated that loss of Lsp1 promotes T cell migration C57BL/6 background, and age-matched and sex- matched into arthritic synovia and draining lymph nodes in mice WT C57BL/6 mice were used as a control. with T cell- dependent chronic inflammation.13 Interest- ingly, several reports have suggested a possible link of Induction of B16 melanoma in mice LSP1 to the pathogenesis of various cancers, including The B16BL6 melanoma cell line (hereafter termed ‘B16 breast cancer,14–16 bladder cancer,17 dermatofibroma18 melanoma’) was purchased from the Korean Cell Line and hepatocellular carcinoma19 20 beyond its role in the Bank (Seoul, Korea). The Lewis lung carcinoma (LLC) migration of immune cells. For example, genetic variation cell line was obtained from the American Type Culture in LSP1 has been implicated in susceptibility, prognostic Collection (Manassas, Virginia, USA). The MC38 colon outcomes and as a diagnostic marker in diverse types adenocarcinoma cell line was kindly provided by Dr of cancers.14–19 21 Moreover, a recent study showed that Tai- Gyu Kim (The Catholic University of Korea, Seoul, high LSP1 levels in glioblastoma serve as an independent Korea). All cell lines were cultured in Dulbecco’s Modi- predictive factor of unfavorable prognosis.22 However, it fied Eagle Medium (Welgene, Gyeongsan, Korea) supple- remains unclear whether LSP1 in T cells directly regu- mented with 10% heat-inactivated fetal bovine serum lates tumor growth and how it contributes to the patho- (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin genesis of cancers. and 0.25 μg/mL Fungizone (Gibco; Thermo Fisher In this study, we postulated that Lsp1 deficiency Scientific, Waltham, Massachusetts, USA). All cell lines promotes the antitumor activity of T cells by inducing used in this study were negative for Mycoplasma, when cell migration and invasion into the tumor mass. We tested using an e- Myco Mycoplasma PCR Detection Kit demonstrated that Lsp1 deficiency in T cells suppresses (iNtRON Biotec, Seongnam, Korea). After being resus- the growth of B16 melanoma in mice, which seems to be pended in phosphate buffered saline (PBS), 5×105 B16 mediated by increased infiltration of CD8+ T cells into melanoma cells, 1×105 MC38 cells or 2.5×105 LLC cells tumor sites and by enhanced production of interferon- were injected subcutaneously into the right flank of mice gamma (IFN-γ) and tumor necrosis factor- alpha (TNF-α), aged 8–12 weeks. Tumor volumes were measured every http://jitc.bmj.com/ antitumor effector cytokines, by T cells. In contrast, Lsp1- 2–3 days with a caliper and calculated according to the overexpressing T cells show the opposite results. Notably, following formula: V(mm3)=D×d2×0.52, where D (mm) Lsp1 KO further potentiates the suppressive effect of anti- and d (mm) are the largest and smallest perpendicular PD-1 Ab on melanoma growth.