PTPN 2 phosphatase deletion in T cells promotes anti‐tumour immunity and CAR T‐cell efficacy in solid tumours
2019; Springer Nature; Volume: 39; Issue: 2 Linguagem: Inglês
10.15252/embj.2019103637
ISSN1460-2075
AutoresFlorian Wiede, Kun‐Hui Lu, Xin Du, Shuwei Liang, Katharina Hochheiser, Garron T. Dodd, Pei Kee Goh, Conor J. Kearney, Déborah Meyran, Paul A. Beavis, Melissa A. Henderson, Simone L. Park, Jason Waithman, Sheng Zhang, Zhong‐Yin Zhang, Jane Oliaro, Thomas Gebhardt, Phillip K. Darcy, Tony Tiganis,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle5 December 2019Open Access PTPN2 phosphatase deletion in T cells promotes anti-tumour immunity and CAR T-cell efficacy in solid tumours Florian Wiede Florian Wiede orcid.org/0000-0001-5145-7180 Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Kun-Hui Lu Kun-Hui Lu Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Xin Du Xin Du Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Shuwei Liang Shuwei Liang Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Katharina Hochheiser Katharina Hochheiser Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Garron T Dodd Garron T Dodd Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Search for more papers by this author Pei K Goh Pei K Goh Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Conor Kearney Conor Kearney Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Deborah Meyran Deborah Meyran Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Paul A Beavis Paul A Beavis Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Melissa A Henderson Melissa A Henderson Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Simone L Park Simone L Park Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Jason Waithman Jason Waithman Telethon Kids Institute, University of Western Australia, Perth, WA, Australia Search for more papers by this author Sheng Zhang Sheng Zhang Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA Search for more papers by this author Zhong-Yin Zhang Zhong-Yin Zhang Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA Search for more papers by this author Jane Oliaro Jane Oliaro Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Thomas Gebhardt Thomas Gebhardt Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Phillip K Darcy Phillip K Darcy Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Tony Tiganis Corresponding Author Tony Tiganis [email protected] [email protected] orcid.org/0000-0002-8065-9942 Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Florian Wiede Florian Wiede orcid.org/0000-0001-5145-7180 Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Kun-Hui Lu Kun-Hui Lu Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Xin Du Xin Du Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Shuwei Liang Shuwei Liang Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Katharina Hochheiser Katharina Hochheiser Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Garron T Dodd Garron T Dodd Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Search for more papers by this author Pei K Goh Pei K Goh Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Conor Kearney Conor Kearney Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Deborah Meyran Deborah Meyran Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Paul A Beavis Paul A Beavis Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Melissa A Henderson Melissa A Henderson Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Simone L Park Simone L Park Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Jason Waithman Jason Waithman Telethon Kids Institute, University of Western Australia, Perth, WA, Australia Search for more papers by this author Sheng Zhang Sheng Zhang Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA Search for more papers by this author Zhong-Yin Zhang Zhong-Yin Zhang Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA Search for more papers by this author Jane Oliaro Jane Oliaro Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Thomas Gebhardt Thomas Gebhardt Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia Search for more papers by this author Phillip K Darcy Phillip K Darcy Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Tony Tiganis Corresponding Author Tony Tiganis [email protected] [email protected] orcid.org/0000-0002-8065-9942 Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia Peter MacCallum Cancer Centre, Melbourne, Vic., Australia Search for more papers by this author Author Information Florian Wiede1,2,3, Kun-Hui Lu3, Xin Du3,4, Shuwei Liang1,2,3, Katharina Hochheiser3,5,6, Garron T Dodd1,2, Pei K Goh1,2,3, Conor Kearney3, Deborah Meyran3, Paul A Beavis3,4, Melissa A Henderson3, Simone L Park5,6, Jason Waithman7, Sheng Zhang8, Zhong-Yin Zhang8, Jane Oliaro3,4, Thomas Gebhardt5,6, Phillip K Darcy3,4 and Tony Tiganis *,*,1,2,3 1Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia 2Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia 3Peter MacCallum Cancer Centre, Melbourne, Vic., Australia 4Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Vic., Australia 5Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic., Australia 6Peter Doherty Institute for Infection and Immunity, Melbourne, Vic., Australia 7Telethon Kids Institute, University of Western Australia, Perth, WA, Australia 8Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Purdue University, West Lafayette, IN, USA *Corresponding author. Tel: +61417396512; E-mails: [email protected]; [email protected] The EMBO Journal (2020)39:e103637https://doi.org/10.15252/embj.2019103637 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8+ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2+ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours. Synopsis This study reveals a role for protein tyrosine phosphatase N2 (PTPN2) in cancer immunosurveillance and T-cell infiltration into solid tumours. Antagonizing PTPN2 thus represents a promising therapeutic strategy for adoptive T-cell therapy. T-cell-specific PTPN2 deletion prevents tumour formation in p53+/− mice without exacerbating inflammation. PTPN2 deletion enhances LCK-mediated CAR T-cell cytotoxicity. PTPN2 deletion promotes STAT5-mediated CXCR3 expression and CAR T-cell homing. PTPN2-deficient CAR T cells eradicate solid tumours in vivo. PTPN2 inhibition enhances the activity of human CAR T cells. Introduction Tumours can avoid the immune system by co-opting immune checkpoints to directly or indirectly inhibit the activation and function of cytotoxic CD8+ T cells (Pardoll, 2012; Ribas & Wolchok, 2018). In particular, the inflammatory tumour microenvironment can upregulate ligands for T-cell inhibitory receptors such as programmed cell death protein-1 (PD-1) on tumour cells to inhibit T-cell signalling and promote the tolerisation or exhaustion of T cells (Pardoll, 2012; Ribas & Wolchok, 2018). Immune checkpoint receptors, including PD-1 and cytotoxic T-lymphocyte antigen-4 (CTLA-4), can suppress the amplitude and/or duration of T-cell responses by recruiting phosphatases to counteract the kinase signalling induced by the T-cell receptor (TCR) and co-stimulatory receptors such as CD28 on αβ T cells (Pardoll, 2012; Zappasodi et al, 2018). Protein tyrosine phosphatase N2 (PTPN2) negatively regulates αβ TCR signalling by dephosphorylating and inactivating the most proximal tyrosine kinase in the TCR signalling cascade, the Src family kinase (SFK) LCK (van Vliet et al, 2005; Wiede et al, 2011). PTPN2 also antagonises cytokine signalling required for T-cell function, homeostasis and differentiation by dephosphorylating and inactivating Janus-activated kinase (JAK)-1 and JAK-3, and their target substrates signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5 in a cell context-dependent manner (ten Hoeve et al, 2002; Simoncic et al, 2002; Wiede et al, 2017a,b). By dephosphorylating LCK, PTPN2 sets the threshold for productive TCR signalling and prevents overt responses to self-antigen in the context of T-cell homeostasis and antigen cross-presentation to establish peripheral T-cell tolerance (Wiede et al, 2014a,b). The importance of PTPN2 in T cells in immune tolerance is highlighted by the development of autoimmunity in aged T-cell-specific PTPN2-deficient mice on an otherwise non-autoimmune C57BL/6 background (Wiede et al, 2011), the systemic inflammation and autoimmunity evident when PTPN2 is deleted in the hematopoietic compartment of adult C57BL/6 mice (Wiede et al, 2017b) and the accelerated onset of type 1 diabetes in T-cell-specific PTPN2-deficient mice on the autoimmune-prone non-obese diabetic (NOD) background (Wiede et al, 2019). In humans, PTPN2 deficiency is accompanied by the development of type 1 diabetes, rheumatoid arthritis and Crohn's disease (Consortium, 2007, Long et al, 2011). The autoimmune phenotype of PTPN2-deficient mice is reminiscent of that evident in mice in which the immune checkpoint receptors PD-1 (Nishimura et al, 1999, 2001; Wang et al, 2005) or CTLA4 (Tivol et al, 1995; Waterhouse et al, 1995) have been deleted. Whole-body PD-1 deletion results in spontaneous lupus-like autoimmunity in C57BL/6 mice (Nishimura et al, 1999) and accelerated type 1 diabetes onset in NOD mice (Wang et al, 2005), whereas CTLA4 deletion in C57BL/6 mice results in marked lymphoproliferation, autoreactivity and early lethality (Tivol et al, 1995; Waterhouse et al, 1995). Although PD-1 and/or CTLA4 blockade can be accompanied by the development of immune-related toxicities, antibodies targeting these receptors have nonetheless shown marked therapeutic efficacy in various tumours, including melanomas, non-small-cell lung carcinomas, renal cancers and Hodgkin lymphoma (Pardoll, 2012; Ribas & Wolchok, 2018). Accordingly, we sought to assess the role of PTPN2 in T-cell-mediated immunosurveillance and the impact of targeting PTPN2 on adoptive T-cell immunotherapy. We especially focused on CAR T-cell therapy, which has shown marked clinical efficacy in B-cell acute lymphoblastic leukaemia (ALL), but has been largely ineffective in solid tumours (Grupp et al, 2013; Maude et al, 2014; Fesnak et al, 2016; Yong et al, 2017). Results PTPN2 deletion prevents tumour formation in p53+/− mice First we determined the impact of deleting PTPN2 in T cells on tumour formation in mice heterozygous for p53, the most commonly mutated tumour suppressor in the human genome (Hollstein et al, 1991). In humans, inheritance of one mutant allele of p53 results in a broad-based cancer predisposition syndrome known as Li-Fraumeni syndrome (Malkin et al, 1990). In mice, p53 heterozygosity results in lymphomas and sarcomas, as well as lung adenocarcinomas and hepatomas in 44% of mice by 17 months of age with the majority of tumours exhibiting p53 loss of heterozygosity (LOH) (Jacks, Jacks et al, 1994). We crossed control (Ptpn2fl/fl) and T-cell-specific PTPN2-null mice (Lck-Cre;Ptpn2fl/fl) onto the p53+/− background and aged the mice for 1 year. Upon necropsy 15/28 (54%), Ptpn2fl/fl;p53+/− mice developed various tumours including thymomas, lymphomas, sarcomas, carcinomas and hepatomas (Fig 1A; Appendix Fig S1; Appendix Table EV1) as reported previously for p53 heterozygous mice (Jacks et al, 1994). In addition, 6/28 mice exhibited splenomegaly accompanied by the accumulation of CD19+IgMhiCD5hiB220int B1 cells consistent with the development of B-cell leukaemias (Fig 1B), whereas CD3-negative CD4+CD8+ double-positive cells reminiscent of T-cell leukaemic blasts (FSC-Ahi) were evident in the thymi or peripheral lymphoid organs of 5/28 mice (Fig 1A and B; Appendix Table EV1). Histological analysis revealed disorganised thymic, lymph node or splenic tissue architecture in diseased Ptpn2fl/fl;p53+/− mice that were predominated by larger lymphoblasts consistent with the accumulation of pre-leukaemic/leukaemic cells (Fig 1B, Appendix Fig S1). By contrast, no Lck-Cre;Ptpn2fl/fl;p53+/− mice (0/22) developed any overt tumours, splenomegaly or abnormal lymphocytic populations as assessed by gross morphology or flow cytometry and lymphoid organ tissue architecture was normal (Fig 1A and B, Appendix Fig S1, Appendix Table EV1). PTPN2 deficiency in T cells can result in inflammation/autoimmunity in aged C57BL/6 mice (Wiede et al, 2011). Accordingly, we determined whether PTPN2 deficiency might exacerbate inflammation in p53+/− mice. We found that inflammation, as assessed by measuring the pro-inflammatory cytokines IL-6, TNF and IFNγ in serum, was elevated in Lck-Cre;Ptpn2fl/fl;p53+/− mice (Appendix Fig S2A), as seen in aged Lck-Cre;Ptpn2fl/fl mice (Appendix Fig S2B), but this did not exceed that occurring in Ptpn2fl/fl;p53+/− littermate controls. Aged Lck-Cre;Ptpn2fl/fl;p53+/− mice also had lymphocytic infiltrates in their livers (Appendix Fig S2C), forming what resembled ectopic lymphoid-like structures (Pitzalis et al, 2014), and this was accompanied by liver damage and ensuing fibrosis (Appendix Fig S2C). However, lymphocytic infiltrates and fibrosis were also evident in the livers of tumour-bearing Ptpn2fl/fl;p53+/− mice (Appendix Fig S2C). Taken together, these results indicate that PTPN2 deficiency in T cells can prevent the formation of tumours induced by p53 LOH without exacerbating inflammation. Figure 1. PTPN2 deletion in T cells increases tumour immunosurveillance A, B. 12-month-old Ptpn2fl/fl;p53+/− and Lck-Cre;Ptpn2fl/f;p53+/− mice were assessed for (A) disease and tumour incidence. (B) Lymphocyte subsets from 12-month-old Ptpn2fl/fl;p53+/− and Lck-Cre;Ptpn2fl/f;p53+/− mice were analysed by flow cytometry. Significance in (A) was determined using two-sided Fisher's exact test. C–F. AT-3-OVA mammary tumour cells were injected into the fourth inguinal mammary fat pads of female Ptpn2fl/fl and Lck-Cre;Ptpn2fl/fl mice and (C) tumour growth monitored over 26 days. (D) At day 26 (d26), the numbers of activated tumour-infiltrating lymphocytes (TILs) were determined. (E) The proportion of IFNγ+ versus IFNγ+TNF+ d26 TILs was determined by flow cytometry. (F) d26 TILs were incubated with AT-3-OVA tumour cells isolated from tumour-bearing C57BL/6 mice, and the proportion of IFNγ+ T cells was determined. Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (C), significance was determined using 2-way ANOVA test and in (D–F) significance determined using 2-tailed Mann–Whitney U-test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint PTPN2 deficiency enhances T-cell-mediated immunosurveillance At least one mechanism by which PTPN2 deficiency might prevent tumour formation in p53+/− mice might be through the promotion of T-cell-mediated tumour immunosurveillance. To explore this, we first assessed the growth of syngeneic tumours arising from ovalbumin (OVA)-expressing AT-3 (AT-3-OVA) mammary carcinoma cells implanted into the inguinal mammary fat pads of Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl C57BL/6 mice (Fig 1C); AT-3 cells lack oestrogen receptor, progesterone receptor and ErbB2 expression and are a model of triple-negative breast cancer (Stewart & Abrams, 2007; Mattarollo et al, 2011). Whereas AT3-OVA cells grew readily in Ptpn2fl/fl mice, tumour growth was markedly repressed in Lck-Cre;Ptpn2fl/fl mice so that tumour progression was prevented in 5/13 mice and eradicated in 2/8 of the remaining mice after tumours had developed. The repression of tumour growth was accompanied by the infiltration of CD4+ and CD8+ effector/memory (CD44hiCD62Llo) T cells into tumours (Fig 1D). Consistent with our previous studies (Wiede et al, 2011), PTPN2-deficient CD25hiFoxP3+ regulatory T cells (Tregs) were increased rather than decreased in AT-3-OVA tumours (Appendix Fig S2D) and their activation was moderately enhanced (Appendix Fig S2E) precluding the repression of tumour growth being due to defective Treg-mediated immunosuppression. Moreover, tumour-infiltrating PTPN2-deficient CD4+ and CD8+ effector/memory T cells were significantly more active, as assessed by the PMA/ionomycin-induced production of markers of T-cell cytotoxicity ex vivo, including interferon (IFN)-γ and tumour necrosis factor (TNF) (Fig 1E). To directly assess the influence of PTPN2 deficiency on T-cell-mediated immunosurveillance, we next isolated tumour-infiltrating CD8+ T cells from Ptpn2fl/fl versus Lck-Cre;Ptpn2fl/fl mice and assessed their activation by measuring IFNγ production ex vivo upon re-challenge with tumour cells isolated from AT3-OVA tumours that had developed in Ptpn2fl/fl mice (Fig 1F). Ptpn2fl/fl tumour-infiltrating CD8+ T cells remained largely unresponsive when re-challenged (Fig 1F), consistent with tolerisation. By contrast, PTPN2-deficient T cells exhibited significant increases in IFNγ consistent with increased effector activity (Fig 1F). These findings point towards PTPN2 having an integral role in T-cell tolerance and immune surveillance. To explore the cellular mechanisms by which PTPN2 deficiency might enhance immunosurveillance, we determined whether PTPN2 deletion might promote the tumour-specific activity of adoptively transferred CD8+ T cells expressing the OT-1 TCR specific for the ovalbumin (OVA) peptide SIINFEKL. Naive OT-1 T cells can undergo clonal expansion and develop effector function when they engage OVA-expressing tumours, but thereon leave the tumour microenvironment, become tolerised and fail to control tumour growth (Shrikant & Mescher, 1999; Shrikant et al, 1999; Thompson et al, 2010). The eradication of solid tumours by naive CD8+ T cells is dependent on help from tumour-specific CD4+ T cells (Marzo et al, 1999; Shrikant et al, 1999). Our previous studies have shown that PTPN2 deficiency enhances TCR-instigated responses and negates the need for CD4+ T-cell help in the context of antigen cross-presentation (Wiede et al, 2014b). Accordingly, we determined whether PTPN2 deficiency might overcome tolerisation and render naive OT-1 CD8+ T cells capable of suppressing the growth of OVA-expressing tumours. To this end, naive OT-1;Ptpn2fl/fl or OT-1;Lck-Cre;Ptpn2fl/fl CD8+ T cells were adoptively transferred into immunocompetent and non-irradiated congenic C57BL/6 hosts bearing syngeneic tumours arising from AT-3-OVA cells inoculated into the mammary fat pad (Fig 2A). As expected (Shrikant & Mescher, 1999; Shrikant et al, 1999), adoptively transferred naive (CD44loCD62Lhi) Ptpn2fl/fl OT-1 CD8+ T cells had no overt effect on the growth of AT-3-OVA mammary tumours when compared to vehicle-treated tumour-bearing mice (Fig 2A). By contrast 5 days after adoptive transfer, Lck-Cre;Ptpn2fl/fl OT-1 T cells completely repressed tumour growth (Fig 2A). The repression of tumour growth was accompanied by an increase in Lck-Cre;Ptpn2fl/fl OT-1 T cells in the draining lymph nodes of the tumour-bearing mammary glands (Appendix Fig S3A) and a marked increase in tumour-infiltrating Lck-Cre;Ptpn2fl/fl OT-1 T cells (Fig 2B; Appendix Fig S3B). At 9 days post-adoptive transfer both tumour and draining lymph node Lck-Cre;Ptpn2fl/fl OT-1 T cells were more active, as assessed by the PMA/ionomycin-induced expression of effector molecules, including IFNγ, TNF and granzyme B (Fig 2C; Appendix Fig S3C). Although the expression of the T-cell inhibitory receptors PD-1 and Lag-3 on tumour-infiltrating PTPN2-deficient OT-1 T cells at 9 days post-transfer was not altered (Appendix Fig S3D), by 21 days post-transfer relative PD-1 and LAG-3 levels were reduced and CD44 was increased on PTPN2-deficient tumour-infiltrating and draining lymph node OT-1 T cells when compared to Ptpn2fl/fl controls (Appendix Fig S3E–G), consistent with decreased T-cell exhaustion. AT3-OVA tumours in mice treated with PTPN2-deficient OT-1 CD8+ T cells started to re-emerge after 21 days, but survival was prolonged for as long as 86 days (Fig 2D; Appendix Fig S3H); by contrast, control mice achieved the maximum ethically permissible tumour burden (200 mm2) by 25 days. Tumour re-emergence in this setting was accompanied by decreased OVA and MHC class I (H2-k1) gene expression, consistent with decreased antigen presentation; tumour re-emergence was also accompanied by decreased PD-L1 (Cd274) gene expression (Fig 2E), but this probably followed decreased MHC class I-mediated antigen presentation and thereby T-cell recruitment and inflammation. Taken together, these results are consistent with PTPN2 deficiency increasing the functional activity and attenuating the tolerisation of naïve CD8+ T cells to suppress tumour growth. Figure 2. PTPN2 deletion enhances CD8+ T-cell-mediated immunosurveillance A–D. AT-3-OVA mammary tumour cells (1 × 106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection, FACS-purified 2 × 106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. Tumour-bearing Ly5.1+ mice were monitored for (A) tumour growth over 21 days and (D) for survival over 86 days. (B) After 21 days, TILs were processed for flow cytometry and donor T-cell numbers (Ly5.1−Ly5.2+) determined. (C) After 9 days, the proportion of Ly5.2+IFNγ+TNF+ versus Ly5.2+GrzB+ TILs was determined. E. Gene expression in tumours from mice treated with Ly5.2+;OT-1;Ptpn2fl/fl T cells 21 days post-adoptive transfer versus those re-emerging in mice treated with Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl T cells. F–I. B16.F10-OVA melanoma cells (1 × 105) were engrafted onto the abraded skin in the flanks of Ly5.1+ mice. 24 h after tumour cell engraftment, naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred and tumour incidence monitored. (G) Epidermal lymphocytes from tumour-free mice were stained for CD69hiCD103hi and donor-derived (Ly5.2+vα2+) tissue-resident memory T cells (TRMs) determined by flow cytometry. (H) Tumour sizes in B16.F10-OVA melanoma bearing mice were determined between days 21 and 23. (I) TILs were assessed for Ly5.2+;OT-1;Ptpn2fl/fl and Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl donor T-cell numbers by flow cytometry. Data information: Representative flow cytometry profiles and results (means ± SEM) from at least two independent experiments are shown. In (A), significance was determined using 2-way ANOVA test and in (B, C, E, G, H, I) significance determined using 2-tailed Mann–Whitney U-test. In (D, F), significance was determined using log-rank (Mantel–Cox) test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Next, we determined whether PTPN2 deficiency might promote T-cell-mediated immunosurveillance and anti-tumour activity in a different tumour setting (Fig 2F–I). Specifically, we utilised an orthotopic transplant model of cutaneous melanoma, where OVA-expressing tumour growth occurs within the epidermis and dermis, mimicking the human condition (Wylie et al, 2015). This model requires that circulating OT-1 CD8+ T cells traffic to the epidermis where they differentiate into non-recirculating CD69+ CD103+ tissue-resident memory T (TRM) cells that contribute to immunosurveillance and the suppression of solid tumour formation (Park et al, 2019). Because of the sporadic nature of tumour development and the protrusion of tumours into the dermal layer (Wylie et al, 2015), we monitored for the number of mice that were macroscopically tumour-free at any one time and measured volumes after resection. We found that 55% of mice receiving PTPN2-deficient OT-1 naive CD8+ T cells were tumour-free at 55 days post-adoptive transfer, whereas only 18% of mice receiving control OT-1 naive CD8+ T cells were tumour-free (Fig 2F) and this was accompanied by the increased presence of OT-1 CD8+ TRMs in the skin (Fig 2G). These findings are consistent with PTPN2 deficiency promoting the differentiation of circulating naive OT-1 T cells into TRMs to prevent tumour formation. Moreover, even where tumours were evident in mice treated with PTPN2-deficient OT-1 T cells, tumour volumes were 10-fold lowe
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