PTPN2 deletion in T cells promotes anti-tumour immunity and CAR T cell efficacy in solid tumours

Although adoptive T cell therapy has shown remarkable clinical efficacy in hematological 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.

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 1,2 . 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 1,2 . 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 ab T cells 2,3 .
Protein tyrosine phosphatase N2 (PTPN2) negatively regulates ab TCR signalling by dephosphorylating and inactivating the most proximal tyrosine kinase in the TCR signalling cascade, the Src family kinase (SFK) LCK 4,5 . 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 [6][7][8][9] . 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 10,11 . 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 nonautoimmune C57BL/6 background 5 , the systemic inflammation and autoimmunity evident when PTPN2 is deleted in the hematopoietic compartment of adult C57BL/6 mice 8 and the accelerated onset of type 1 diabetes in T cell-specific PTPN2-deficient mice on the autoimmune-prone non-obese diabetic (NOD) background 12 . In humans, PTPN2 deficiency is accompanied by the development of type 1 diabetes, rheumatoid arthritis and Crohn's disease 13,14 . The autoimmune phenotype of PTPN2-deficient mice is reminiscent of that evident in mice in which the immune checkpoint receptors PD-1 [15][16][17] or CTLA-4 18,19 have been deleted. Whole-body PD-1 deletion results in spontaneous lupus-like autoimmunity in C57BL/6 mice 15 and accelerated type 1 diabetes onset in NOD mice 17 , whereas CTLA4 deletion in C57BL/6 mice results in marked lymphoproliferation, autoreactivity and early lethality 18,19 . 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 1,2 . 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 leukemia (ALL), but has been largely ineffective in solid tumours [20][21][22][23] .

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 24 . In humans, inheritance of one mutant allele of p53 results in a broad-based cancer predisposition syndrome known as Li-Fraumeni syndrome 25 . 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) 26 . We crossed control (Ptpn2 fl/fl ) and T cell-specific PTPN2-null mice (Lck-Cre;Ptpn2 fl/fl ) onto the p53 +/background and aged the mice for one year. Upon necropsy 15/28 (54%) Ptpn2 fl/fl ;p53 +/mice developed various tumours including thymomas, lymphomas, sarcomas, carcinomas and hepatomas ( Fig. 1a; Fig. S1; Table S1) as reported previously for p53 heterozygous mice 26 . In addition, 6/28 mice exhibited splenomegaly accompanied by the accumulation of CD19 + IgM hi CD5 hi B220 int B1 cells consistent with the development of B cell leukemias (Fig. 1b), whereas CD3 negative CD4+CD8+ double positive cells reminiscent of T cell leukemic blasts (FSC-A hi ) were evident in the thymi or peripheral lymphoid organs of 5/28 mice ( Fig. 1a-b; Table S1).
Histological analysis revealed disorganised thymic, lymph node or splenic tissue architecture in diseased Ptpn2 fl/fl ;p53 +/mice that was predominated by larger lymphoblasts consistent with the accumulation of pre-leukemic/leukemic cells (Fig.   S1). By contrast no Lck-Cre;Ptpn2 fl/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. 1; Fig. S1).
PTPN2 deficiency in T cells can result in inflammation/autoimmunity in aged C57BL/6 mice 5 . Accordingly, we determined if PTPN2-deficiency might exacerbate inflammation in p53 +/mice. We found that inflammation, as assessed by measuring the proinflammatory cytokines IL-6, TNF and IFNg in plasma, was elevated in Lck-Cre;Ptpn2 fl/fl ;p53 +/mice (Fig. S2a), as seen in aged Lck-Cre;Ptpn2 fl/fl mice (Fig. S2b), but this did not exceed that occurring in Ptpn2 fl/fl ;p53 +/littermate controls. Aged Lck-Cre;Ptpn2 fl/fl ;p53 +/mice also had lymphocytic infiltrates in their livers (Fig. S2c), forming what resembled ectopic lymphoid-like structures 27 and this was accompanied by liver damage and ensuing fibrosis (Fig. S2c). However lymphocytic infiltrates and fibrosis were also evident in the livers of tumour-bearing Ptpn2 fl/fl ;p53 +/mice ( Fig.   S2c). Taken together results indicate that PTPN2 deficiency in T cells can prevent the formation of tumours induced by p53 LOH without exacerbating inflammation.

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) adenocarcinoma cells implanted into the inguinal mammary fat pads of Ptpn2 fl/fl versus Lck-Cre;Ptpn2 fl/fl C57BL/6 mice (Fig. 1c); AT-3 cells lack estrogen receptor, progesterone receptor and ErbB2 expression and are a model of triple negative breast cancer 28 29 . Whereas AT3-OVA cells grew readily in Ptpn2 fl/fl mice, tumour growth was markedly repressed in Lck-Cre;Ptpn2 fl/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 (CD44 hi CD62L lo ) T cells into tumours (Fig. 1d). Consistent with our previous studies 5 , PTPN2-deficient CD25 hi FoxP3 + regulatory T cells (Tregs) were increased rather than decreased in AT3-OVA tumours (Fig. S2d) and their activation was increased (data not shown) precluding the repression of tumour growth being due to defective Treg-mediated immunosuppression. Moreover, tumour-infiltrating PTPN2deficient 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) g and tumour necrosis factor (TNF) (Fig. 1e); by contrast splenic PTPN2-deficient T cells exhibited comparatively small increases in cytotoxic capacity as assessed by IFNg/TNF expression (data not shown). To directly assess the influence of PTPN2-deficiency on T cell-mediated immunosurveillance, we next isolated tumour-infiltrating CD8+ T cells from Ptpn2 fl/fl versus Lck-Cre;Ptpn2 fl/fl mice and assessed their activation by measuring IFNg production ex vivo upon rechallenge with tumour cells isolated from AT3-OVA tumours that had developed in Ptpn2 fl/fl mice (Fig. 1f). Ptpn2 fl/fl tumour-infiltrating CD8+ T cells remained largely unresponsive when re-challenged, consistent tolerisation. By contrast, PTPN2-deficient T cells exhibited significant increases in IFNg and TNF consistent with increased effector activity (Fig. 1f). These findings point towards PTPN2 having an integral role in T cell-mediated immune surveillance.
To explore the cellular mechanisms by which PTPN2-deficiency might influence immunosurveillance, we determined whether PTPN2 deletion might promote the tumour-specific activity of adoptively transferred TCR transgenic 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 [30][31][32] . The eradication of solid tumours by naive CD8+ T cells is dependent on help from tumour-specific CD4+ T cells 31,33 . Our previous studies have shown that PTPN2-deficiency enhances TCRinstigated responses and negates the need for CD4+ T cell help in the context of antigen cross-presentation 11 . 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;Ptpn2 fl/fl or OT-1;Lck-Cre;Ptpn2 fl/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 30,31 adoptively transferred naive (CD44 lo CD62L hi ) Ptpn2 fl/fl OT-1 CD8+ T cells (Gating strategy; Fig.   S3) 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;Ptpn2 fl/fl OT-1 T cells completely repressed tumour growth ( Fig. 2a). The repression of tumour growth was accompanied by an increase in Lck-Cre;Ptpn2 fl/fl OT-1 T cells in the draining lymph nodes of the tumour-bearing mammary glands (Fig. S4a) and a marked increase in tumour-infiltrating Lck-Cre;Ptpn2 fl/fl OT-1 T cells ( Fig. 2b; Fig. S4b). At 9 days post adoptive transfer both tumour and draining lymph node Lck-Cre;Ptpn2 fl/fl OT-1 T cells were more active, as assessed by the PMA/ionomycin-induced expression of effector molecules, including IFNg, TNF and granzyme B ( Fig. 2c; Fig. S4c). 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 (Fig. S4d), by 21 days post-transfer relative PD-1 and LAG-3 levels were reduced and CD44 was increased on PTPN2-deficient tumourinfiltrating and draining lymph node OT-1 T cells when compared to Ptpn2 fl/fl controls ( Fig. S4e-g), consistent with the possibility of decreased T cell exhaustion. AT3-OVA tumours in mice treated with PTPN2-deficent OT-1 CD8+ T cells started to re-emerge after 21 days, but survival was prolonged for as long as 86 days ( Fig. 2d; Fig. S4h); by contrast control mice achieved the maximum ethically permissible tumour burden (200 mm 2 ) 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 antigenpresentation; tumour re-emergence was also accompanied by decreased PD-L1 (Cd274) gene expression (Fig. 2e), but this probably followed decreased MHC class Imediated 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.

PTPN2-deficiency enhances CAR T cell cytotoxicity
CAR T cells are autologous T cells engineered to express a transmembrane CAR specific for a defined tumour antigen that signals via canonical TCR signalling intermediates such as LCK 23,34 . CAR T cells targeting CD19 have especially been impressive in the treatment of ALL, with clinical response rates of up to 90% in pediatric B cell ALL patients 20,21 . However, therapeutic efficacies of CAR T cells in other malignancies, including solid tumours, have been relatively poor 22,23 . Given our findings on PTPN2 in T cell mediated immunosurveillance and anti-tumour immunity, we determined whether targeting PTPN2 might enhance the function of CAR T cells in solid tumours. In particular, we assessed the therapeutic efficacy of second-generation CAR T cells harboring the intracellular signalling domains of CD28 and CD3z and targeting the human orthologue of murine ErbB2/Neu, HER-2 35 . HER-2 is overexpressed in many solid tumours, including 20% of breast cancers, where it promotes tumour aggressiveness and metastasis 36 .

First, we assessed the impact of PTPN2 deletion on CAR T cells in vitro.
Consistent with PTPN2's role in setting thresholds for TCR-instigated responses 5,10,11 , we found that PTPN2-deficiency resulted in ten-fold lower concentrations of TCR crosslinking antibodies (a-CD3e) being required for the maximal generation of CD8 + HER-2 CAR T cells in vitro, with the resulting CAR T cells being predominated by the effector/memory (CD44 hi CD62L lo ) subset (Fig. S5a). Next, we assessed the impact of PTPN2-deficiency on antigen-induced CAR T cell activation in vitro. PTPN2-deficient CD8 + HER-2 CAR T cells were more activated, as assessed by the expression of CD44, CD25, PD-1 and LAG-3, after overnight incubation with HER-2-expressing 24JK (24JK-HER-2) sarcoma cells, but importantly, not HER-2-negative 24JK control cells ( Fig. S5b). Moreover, PTPN2-deficient CD8 + HER-2 CAR T cells exhibited increased antigen-specific cytotoxic capacity in vitro ( Fig. S5c; Fig. S6a), as assessed by the increased intracellular expression of IFNg (required for tumour eradication by CAR T cells in vivo 37 ), TNF and granzyme B upon challenge with 24JK-HER-2 cells but not 24JK cells. Moreover, both effector/memory and central memory (CD44 hi CD62L hi ) PTPN2-deficient CD8+ HER-2 CAR T cells were more effective at specifically killing 24JK-HER-2 cells but not 24JK cells in vitro (Fig. S6b). Taken together these results are consistent with PTPN2-deficiency enhancing not only the generation, but also the antigen-specific activation and cytotoxicity of CAR T cells in vitro.

PTPN2-deficiency enhances LCK-dependent CAR T cell function
Next, we explored the mechanisms by which PTPN2-deficiency may influence CAR T cell activation and function. PTPN2 dephosphorylates and inactivates the SFK LCK to tune TCR signalling so that T cells can differentially respond to self versus non-self 5,[9][10][11] . CAR T cells are reliant on canonical TCR signalling intermediates, including LCK for their activation and function 34 . Accordingly, we assessed the influence of PTPN2 deficiency on the activation of LCK in CAR T cells by monitoring for the phosphorylation of Y394 (using antibodies specific for Y418-phosphorylated SFKs). PTPN2-deficiency significantly increased SFK Y418 phosphorylation in CD8+ HER-2 CAR T cells ( Fig. S7a; Fig. 3a). We have shown previously that the enhanced TCR-induced T cell activation resulting from PTPN2 deficiency is accompanied by the increased expression of the interleukin (IL)-2 receptor chains CD25 (IL-2 receptor a chain) and CD122 (IL-2/15 receptor b chain) and IL-2 induced STAT-5 signalling 5,11 .
Consistent with this we found that CD25, CD122 and CD132 (common g chain shared by IL-2 and IL-15) receptor levels were elevated in activated PTPN2-deficient CAR T cells ( Fig. S7b; Fig. 3b) and this was accompanied by increased basal and IL-2/15induced STAT-5 Y694 phosphorylation ( Fig. S7c-d). To explore the extent to which the increased SFK signalling may contribute to this and the enhanced CAR T cell activation and function we crossed Lck-Cre;Ptpn2 fl/fl mice onto the Lck +/background 9 so that total LCK would be reduced by 50% and LCK signalling may more closely approximate that in Ptpn2 fl/fl controls. Consistent with this we found that SFK Y418 phosphorylation in Lck-Cre;Ptpn2 fl/fl ;Lck +/-CD8+ HER-2 CAR T cells was reduced to that in Ptpn2 fl/fl controls (Fig. 3a). Strikingly, Lck heterozygosity attenuated the enhanced antigen-specific HER-2 CAR T cell activation (as monitored by CD44, CD25, PD-1 and LAG-3 levels; Fig. 3b) and cytotoxic potential (as measured by the antigen-induced expression of IFNg and TNF; Fig. 3c) and the enhanced capacity of PTPN2-deficient CAR T cells to specifically kill HER-2-expressing tumour cells (Fig.   3d). In addition, Lck heterozygosity corrected the enhanced IL-2/15 receptor levels ( Fig. 3e; Fig. S7e) and partially corrected the enhanced IL-2/15-induced STAT-5 signalling (Fig. S7f). The persistent increased STAT-5 signalling despite correcting IL-2/15 receptor levels is consistent with previous studies showing that STAT-5 can also serve as direct a bona-fide substrate of PTPN2 and that PTPN2-deficiency promotes cytokine-induced STAT-5 signalling in thymocytes/T cells 5,6,9,10,38,39 . Irrespective, these results are consistent with PTPN2 deficiency enhancing the antigen-specific activation and function of CAR T cells through the promotion of LCK signalling.

PTPN2-deficient CAR T cells eradicate solid tumours
To explore the therapeutic efficacy of PTPN2-deficient HER-2-targeting CAR T cells in vivo we adoptively transferred a single dose (6 x 10 6 ) of purified Ptpn2 fl/fl versus Lck-Cre;Ptpn2 fl/fl central memory CD8+ HER-2 CAR T cells into sub-lethally irradiated syngeneic recipients bearing established orthotopic tumours arising from the injection of HER-2-expressing E0771 (HER-2-E0771) breast cancer cells (Fig. 4). We adoptively transferred central memory CAR T cells as these cells engraft better and elicit persistent anti-tumour responses 40 . In addition, as lymphodepletion prior to T cell infusion is used routinely in the clinic to facilitate T cell expansion and enhance efficacy, 41 we immunodepleted mice by sublethal irradiation (400 cGy) as is routine in murine CAR T cell studies 42 . Notably, HER-2-E0771 cells were grafted into HER-2 transgenic (TG) mice, where HER-2 expression was driven by the whey acidic protein (WAP) promoter that induces expression in the cerebellum and the lactating mammary gland 43 , so that HER-2-expressing orthotopic tumours would be regarded as self and host anti-tumour immunity repressed. Previous studies have shown that effective tumour killing and eradication by CD8+ CAR T cells is reliant on the presence of CD4+ CAR T cells 37 . Strikingly, we found that although PTPN2-expressing central memory CD8+ HER-2 CAR T cells modestly suppressed HER-2-E0771 mammary tumour growth, PTPN2-deficient CD8+ HER-2 CAR T cells eradicated tumours (Fig. 4a). The ability of PTPN2-deficient CAR T cells to suppress tumour growth and eradicate tumours was reliant on the enhanced activation of LCK, as this was significantly, albeit not completely abrogated by Lck heterozygosity (Fig. 4b) that corrected the enhanced LCK activation in Lck-Cre;Ptpn2 fl/fl CAR T cells in vitro (Fig. 3a). The repression of tumour growth by PTPN2-deficient CD8+ HER-2 CAR T cells occurred despite tumours harboring an immunosuppressive microenvironment 44,45 , with increased immunosuppressive myeloid-derived suppressor cells (MDSCs) (Fig. 4c) and Tregs ( Fig. 4d) and the increased expression of immunosuppressive cytokines, including transforming growth factor b (Tgfb) and IL-10 (Il10) (Fig. 4e), when compared to normal mammary tissue. PTPN2-deficient CAR T cells markedly suppressed tumour growth, even when the tumours were allowed to grow to one quarter (50 mm 2 ) of the maximal ethically permissible mammary tumour burden prior to CAR T cell therapy ( Fig. 4f). Moreover, PTPN2-deficient CAR T cells were effective in repressing tumour growth even without the co-administration of IL-2 that is used routinely in rodent preclinical models to promote CAR T cell expansion, but is not used in the clinic (Fig. 4g).
Taken together these results demonstrate that PTPN2-deficiency promotes the LCKdependent activation of CAR T cells and overcomes the immunosuppressive tumour microenvironment to eradicate solid tumours in vivo.
Strikingly the repression of tumour growth by PTPN2-deficient HER-2 CAR T cells persisted long after HER-2 tumours had been eradicated, so that approximately half of all mice were alive for longer than 200 days with the remaining half succumbing to tumours (Fig. 5a). Moreover, the tumours that did re-emerge lacked HER-2 as assessed by immunohistochemistry (Fig. 5b) and quantitative real time PCR (Fig. 5c).
These results are consistent with PTPN2-deficient HER-2 CAR T cells completely eliminating HER-2 expressing tumours and eliciting a selective pressure so that any reemerging tumours downregulate HER-2. To explore this further and the influence of PTPN2-deficiency on CAR T cell memory, we re-implanted HER-2+ tumour cells into control HER-2 transgenic mice, or those mice in which HER-2+ tumours had been previously cleared by PTPN2-deficient HER-2 CAR T cells (Fig. 5d-e). In those mice in which tumours had previously been cleared, splenic PTPN2-deficient CAR T cells had a central memory phenotype (CD44 hi CD62L hi ) rather than the mixed central and effector/memory (CD44 hi CD62L lo ) phenotypes otherwise present on day 10 post adoptive transfer ( Fig. 5f-g), consistent with PTPN2 deficiency promoting CAR T cell memory. To assess systemic anti-tumour immunity, HER-2+ tumours cells were reimplanted into the previously tumour-free contralateral mammary fat pads. As a control, we also monitored the growth of HER-2-negative E0771 tumour cells. We found that the growth of HER-2+ tumours in the previously tumour-free contralateral mammary fat pads was markedly repressed or completely prevented (Fig. 5d). The repression of tumour growth was accompanied by the increased infiltration of HER-2 CAR T cells (Fig. 5e). By contrast, the growth of HER-2-negative mammary E0771 tumours was not affected (Fig. 5d). These results are consistent with PTPN2-deficiency in HER-2 CAR T cells promoting CAR T cell memory and recall to prevent the reemergence of HER-2+ tumours, including those that may arise at distant metastatic sites.

PTPN2-deficiency promotes CAR T cell homing
The clinical benefit of adoptive T cell therapy is highly reliant on the ability of T cells including CAR T cells to home efficiently into the target tissue 46,47 . We found that the repression of tumour growth by PTPN2-deficient CD8+ HER-2 CAR T cells was accompanied by a marked increase in CD45 + CD8 + CD3 + mCherry + CAR T cell abundance in tumours and the corresponding draining lymph nodes (Fig. 6a). In part, the increased CAR T cell abundance may reflect the expansion of CAR T cells after they engage tumour antigen, as PTPN2-deficiency increased the antigen-specific proliferation of HER-2 CAR T cell in vitro ( Fig. 6b; Fig. S8a). However, the increased CAR T cell abundance may also reflect an increase in CAR T cell homing and infiltration as CD3e+ lymphocytes accumulated within the tumour and at the tumour/stromal interface at 10 days post adoptive transfer (Fig. S8b). Previous studies have correlated the accumulation of TILs in tumours with the expression of the chemokine receptor CXCR3 (receptor for CXCL9, CXCL10 and CXCL11) 46,47 . We found that PTPN2-deficient HER-2 CAR T cells expressed higher cell surface levels of the chemokine receptor CXCR3. By contrast, cell surface levels of other chemokine receptors, including CXCR5, CCR7 and CCR5 (receptor for CCL3, CCL4 and CCL5), were not altered ( Fig. 6c-d; Fig. S8c). The ligands for CXCR3 are increased in many tumours and associated with the intralesional accumulation of TILs and improved outcome 46,47 and Cxcl9 and Cxcl10 (Cxcl11 is not expressed in C57BL/6 mice 48 ) were elevated in the HER-2-E0771 tumours analysed at 10 days after implantation (Fig. 6e).
Moreover, consistent with the potential for increased homing, we found that PTPN2deficient CXCR3 hi CAR T cells accumulated in HER-2-E0771 tumours within 3 days of adoptive transfer (Fig. 6f), prior to any effects on tumour burden (Fig. 4a).
To explore whether the increased cell surface CXCR3 might contribute to the increased homing and anti-tumour activity of PTPN2-deficient CAR T cells, we sought to correct the increased CXCR3 expression. CXCR3 is not detected in naïve T cells but is abundant in CD4+ TH1 cells and CD8+ cytotoxic T lymphocytes (CTLs). When CD8+ T cells are activated by a strong TCR stimulus and subsequently stimulated with IL-2 they undergo differentiation into effectors and acquire CTL activity characterized by IFNg and granzyme B expression 49,50 . Our previous studies have shown that PTPN2-deficiency enhances the IL-2-induced generation of effectors from TCR crosslinked and activated T cells 11 . Given that CAR T cell generation is reliant on TCR crosslinking (a-CD3e/a-CD8) and stimulation with IL-2 and IL-7, we determined whether the enhanced LCK activation and increased downstream IL-2 receptor and STAT-5 signalling in PTPN2-deficient CAR T cells might be responsible for the increased CXCR3 expression. To assess this we took advantage of PTPN2-deficient CD8+ CAR T cells that were heterozygous for Lck (Lck-Cre;Ptpn2 fl/fl ;Lck +/-). Since IL-2-induced STAT-5 signalling was only partially corrected in Lck-Cre;Ptpn2 fl/fl ;Lck +/-CAR T cells (Fig. S7f) we also crossed the Lck-Cre;Ptpn2 fl/fl mice onto the Stat5 fl/+ background so that we could independently correct the increased STAT-5 signalling ( Fig. 6g-h). Cell surface CXCR3 levels were significantly albeit modestly reduced in Lck heterozygous CAR T cells (Fig. 6g). By contrast, Stat5 heterozygosity completely corrected the enhanced IL-2 and IL-15-induced STAT-5 signalling and almost completely corrected the increased CXCR3 ( Fig. 6g-

h).
Although CXCR3 is not transcribed by STAT-5, there is evidence that STAT-5 can drive the expression of the transcription factor T-bet (T-box transcription factors T-box expressed in T cells) 51 which together with Eomes (eomesodermin) dictates CD8+ T cell differentiation and function. Indeed T-bet can drive the expression of CXCR3 52 and has been shown to be important for the infiltration and anti-tumour activity of cytotoxic CD8+ T cells 53 . Consistent with this we found that PTPN2deficiency in CAR T cells was associated with increased intracellular T-bet expression that was corrected by Stat5 heterozygosity (Fig. S8d-e). Strikingly, Stat5 but not Lck heterozygosity prevented the increased homing of PTPN2-deficient CAR T cells evident at 3 days post adoptive transfer (Fig. 7a) and largely, albeit not completely, attenuated the ability of PTPN2-deficent CAR T cells to suppress the growth solid tumours (Fig. 7b). The repression of CAR T cell infiltration was also evident in resected tumours at day 16 with Lck-Cre;Ptpn2 fl/fl ;Stat5 fl/+ CAR T cell cytotoxicity markers (TNF, IFNg; induced by PMA/ionomycin ex vivo) being reduced to those in Ptpn2 fl/fl control CAR T cells (Fig. 7c). Therefore, the promotion of STAT-5 signalling might not only promote CXCR3 expression and the homing of CAR T cells to CXCL9/10expressing tumours, but also contribute to the acquisition of CTL activity probably through the induction of T-bet and thereby CAR T cell function.
To complement these findings and further explore the extent to which the CXCR3/CXCL9/10/11 axis might contribute to the increased homing and efficacy of PTPN2-deficient CAR T cells we sought to repress the Cxcl9/10 expression in HER-2-E0771 mammary tumours and assess the impact on the homing and function of PTPN2deficient CAR T cells. As Cxcl9/10 are transcriptional targets of STAT-1 and PTPN2 dephosphorylates STAT-1 to repress IFNg-induced STAT-1-mediated transcription 7,38,54,55 , we generated HER-2-E0771 cells in which PTPN2 could be inducibly overexpressed in response to doxycycline (Fig. 7d). The inducible overexpression of PTPN2 not only repressed IFNg-induced p-STAT-1 (Fig. 7d) and Cxcl9/10 expression ( Fig. 7e), but most importantly, also the recruitment of PTPN2-deficient CAR T cells to HER-2-E0771 mammary tumours in vivo ( Fig. 7f-h). Importantly, although PTPN2deficient CAR T cells remained more effective than controls, the doxycycline-inducible overexpression of PTPN2 in HER-2-E0771 cells and the decreased CAR T cell recruitment attenuated the repression of tumour growth and prevented the eradication of tumours (Fig. 7g-h). Taken together our findings are consistent with the efficacy of PTPN2-deficient CAR T cells in solid tumours being attributed to i) the increased LCKdependent activation of CAR T cells after antigen engagement, ii) the LCK and STAT-5-dependent acquisition of CTL activity and iii) the increased STAT-5 mediated and CXCR3-dependent homing of PTPN2-deficient CAR T cells to CXCL9/10-expressing tumours.

PTPN2-deficient CAR T cells do not promote morbidity
A potential complication of enhancing the function of CAR T cells is the development of systemic inflammation and autoimmunity 23,56 . We found that the eradication of tumours by PTPN2-deficient HER-2 CAR T cells was not accompanied by systemic T cell activation and inflammation, as assessed by the unaltered number and activation of T cells in lymphoid organs at 21 days post-transfer (Fig. S9a) and the unaltered circulating pro-inflammatory cytokines (Fig. S9b) and lymphocytic infiltrates in non-lymphoid tissues, including in the contralateral tumour-negative mammary glands, lungs and livers ( Fig. S9c; data not shown). To further explore any impact of PTPN2-deficiency on the development of inflammatory disease, we increased the number of adoptively transferred CAR T cells from 6 x 10 6 to 20 x 10 6 and monitored for inflammation and autoimmunity. The increased CAR T cell numbers resulted in a more profound repression of tumour growth irrespective of PTPN2 status but only PTPN2-deficient CAR T cells eradicated tumours (Fig. S9d). PTPN2deficiency did not exacerbate systemic inflammation, as assessed by monitoring for lymphocytic infiltrates in non-lymphoid tissues, including the contralateral mammary glands (data not shown), lungs and livers (Fig. S9e) or for circulating IL-6, IFNg, TNF and IL-10 over time (Fig. S10a). Although PTPN2-deficient CAR T cells increased core temperature as early as 5 days post adoptive transfer (Fig. S10b), this is to be expected for a developing immune response and this did not persist after tumours were cleared and did not affect body weight (Fig. S10b-c). In addition, the increased PTPN2deficient CAR T cells did not result in autoimmunity as reflected by the absence of circulating anti-nuclear antibodies (Fig. S10d) and the lack of any overt tissue damage, including liver damage, as assessed by measuring the liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST) in serum (Fig. S10e). Therefore, PTPN2-deficient CAR T cells eradicate tumours without promoting systemic inflammation and immunopathologies.
Another potential complication of CAR T cell therapy is 'on-target off-tumour' toxicities 23,56 . Previous studies have shown that the WAP promoter in the HER-2 TG mice drives HER-2 in the lactating mammary gland and the cerebellum 43 . Although PTPN2-deficient CAR T cells were not evident in the contralateral tumour-negative mammary glands, this is not unexpected as we used virgin mice in our studies. By contrast the WAP promoter 43 drives HER-2 expression in the cerebellum and we could detect HER-2 throughout the cerebellum (Fig. S11a) to similar levels seen in HER-2-E0771 mammary tumours (Fig. S11b). Although we detected some CD3 + mCherry + CAR T cells surrounding the crus 1 of the ansiform lobule of the cerebelum they were not detected in other regions (Fig. S11c) and was there no evidence for overt cerebellar tissue damage (Fig. S11d), as assessed on day 10 post-adoptive transfer when PTPN2deficient CAR T cells were activated (Fig. S11e). Consistent with this, PTPN2deficient CAR T cells did not result in overt morbidity up to 50 days post-transfer and did not affect the cerebellar control of neuromotor function, as assessed in rotarod tests ( Fig. S11f), even in those mice were a greater number of CAR T cells were adoptively transferred (data not shown). In part, the lack of toxicity may be due to CXCR3expressing CAR T cells being unable to efficiently home and infiltrate into the cerebellum, as the cerebellum expressed negligible levels of Cxcl9 and Cxcl10 when compared to the HER-2-E0771 tumours (Fig. S11g). Therefore, targeting PTPN2 may not only enhance the antigen-specific activation and function of CAR T cells, but also limit 'on-target off-tumour' toxicities by driving the homing of CXCR3-expressing CAR T cells to CXCL9/10/11-expressing tumours. Taken together, our results demonstrate that PTPN2 deletion in murine CD8+ CAR T cells dramatically enhances their recruitment into the tumour site, as well as their antigen-specific activation and ability to overcome the immunosuppressive tumour microenvironment to effectively suppress the growth of solid tumours without promoting overt morbidity.

PTPN2 inhibition enhances the antigen-specific activation of human CAR T cells
To explore whether targeting PTPN2 in human T cells and CAR T cells might similarly promote their activation and function we took advantage of a highly specific PTPN2 active site inhibitor, compound 8 58,59 . Treatment of murine CD8+ HER-2 CAR T cells with compound 8 in vitro increased their antigen-specific cytotoxic potential to levels seen in Lck-Cre;Ptpn2 fl/fl HER-2 CAR T cells, but had no additional effect on Lck-Cre;Ptpn2 fl/fl HER-2 CAR T cells, consistent with the inhibitor acting specifically to inhibit PTPN2 and thereby activate CAR T cells upon engagement with specific tumour antigen (Fig. S12a). Importantly, we found that treatment of human peripheral blood mononuclear cells with compound 8 enhanced their activation (as assessed by the activation marker CD69) and the expansion of CD8 + CCR7 + CD45RA + CD69 + T cells in response to TCR ligation (a-CD3e) (Fig. S12b). To assess whether targeting of PTPN2 might enhance the cytotoxic potential of human CAR T cells we took advantage of human CAR T cells targeting the Lewis Y (LY) antigen 60,61 that is overexpressed in many human cancers, including 80% of lung adenocarcinomas, 25% of ovarian carcinomas and 25% of colorectal adenocarcinomas (Fig. 8a). Treatment of human LY CAR T cells with compound 8 significantly enhanced their cytotoxic potential, as assessed by the expression of IFNg and TNF, in response to CAR crosslinking (with a-LY) or engagement with LY-expressing human ovarian (OVCAR-3) carcinoma cells, but not breast cancer cells (MDA-MB-231) that do not express LY (Fig. 8a). Taken together these results are consistent with PTPN2 targeting increasing the potential therapeutic efficacy of human CAR T cells as seen in our pre-clinical models.

PTPN2 knockdown or deletion enhances the therapeutic efficacy of CAR T cells
Whole-body, T cell-or hematopoietic compartment-specific PTPN2 deletion in mice results in systemic inflammation, overt autoreactivity and morbidity 5,8,12,62,63 . The potential for inflammatory complications, including cytokine release syndrome, preclude the utility of PTPN2 inhibitors for systemic therapy and the promotion of T cell mediated anti-tumour immunity. Accordingly, we sought alternate ways by which to target PTPN2 in the context of adoptive cell therapy. To this end we first knocked down Ptpn2 in murine CAR T cells by RNA interference using nuclease resistant siRNA duplexes (siSTABLE™) that efficiently knock down genes for prolonged periods ( Fig. 8b-

d, Fig. S12c-f). HER-2 CAR T cells were transfected with GFP-or
Ptpn2-specific siSTABLE™ siRNAs two days prior to adoptive cell therapy; PTPN2 was knocked down in approximately one third of total HER-2 CAR T cells as assessed by flow cytometry (Fig. 8b) using validated antibodies 10 . Ptpn2 knockdown enhanced the tumour antigen-specific activation/cytotoxic potential and killing capacity of HER-2 CAR T cells ex vivo ( Fig. S12c-e) and markedly repressed the growth of HER-2-E0771 mammary tumours in vivo (Fig. 8c). The repression of tumour growth was accompanied by the significant infiltration of mCherry+ CD8+ HER-2 CAR T cells into tumours (Fig. 8d); infiltrating HER-2 T cells exhibited increased cytotoxic capacity (as assessed by IFNg and TNF expression after PMA/ionomycin treatment ex vivo) (Fig. S12f). By contrast mCherry+ CD8+ HER-2 CAR T cell numbers in the spleen were not affected by Ptpn2 knockdown (Fig. 8d). Therefore, the transient repression of PTPN2, even in a fraction of adoptively transferred CAR T cells, is sufficient to significantly enhance their activity/cytotoxicity and efficacy in vivo.
An alternate approach by which to target PTPN2 in T cells is through CRISPR-Cas9 genome-editing 64 . In particular, we took advantage of Cas9 ribonucleoprotein (RNP)-mediated gene-editing to effectively delete PTPN2 in CAR T cells. This plasmid-free approach allows for efficient but transient genome editing ex vivo so that resultant CAR T cells do not overexpress Cas9 and do not elicit immunogenic responses post-adoptive transfer. To this end we transfected total CAR T cells with recombinant nuclear localized Cas9 pre-complexed with short guide (sg) RNAs capable of directing Cas9 to the Ptpn2 locus ( Fig. 8e-g; Fig. S12g). sgRNAs targeting the Ptpn2 locus completely ablated PTPN2 protein in HER-2 CAR T cells (Fig. 8e) and enhanced their antigen-specific activation/cytotoxic potential (assessed by IFNg production) and their capacity to specifically kill HER-2 expressing 24JK cells ex vivo (Fig. S12g).
Importantly we found that Ptpn2 deletion increased the infiltration of mCherry+ HER-2 CAR T cells into HER-2-E0771 mammary tumours (Fig. 8f) and led to the effective eradication of HER-2-E0771 mammary tumours (Fig. 8g) in vivo. These results demonstrate that CRISPR-Cas9 genome editing can be used to efficiently ablate PTPN2 to enhance the therapeutic efficacy of CAR T cells in solid cancer.

DISCUSSION
Approaches aimed at harnessing host immunity to destroy tumour cells have revolutionized cancer therapy 1,2 . Such approaches have relied on the targeting of immune checkpoints, such as PD-1, to alleviate inhibitory constraints on T cellmediated anti-tumour immunity in immunogenic tumours, or alternatively on adoptive T cell therapy, especially that employing CAR T cells 1,2,23 . Although the latter does not require pre-existing anti-tumour immunity, there are significant hurdles limiting efficacy and prohibiting the widespread utility of CAR T cells in the treatment of solid tumours 23 . These include inefficient CAR T cell homing and infiltration into solid tumours and inadequate CAR T cell activation in the solid tumour microenvironment, which can be overtly immunosuppressive 23 . Like PD-1 15,17 , PTPN2 is fundamentally important in mediating T cell tolerance in mice and humans 5,[10][11][12][13][14] . This is underscored by the striking phenotype similarities between PTPN2-deficient mice and those null for PD-1 5,12,15,17,62,63 . Consistent with this, our studies herein demonstrate that the deletion of PTPN2 in T cells enhances cancer immunosurveillance and the anti-tumour activity of adoptively transferred T cells. In particular, our studies demonstrate that the deletion of PTPN2 not only drives the homing of CAR T cells to solid tumours, but also their activation to eradicate tumours in an otherwise immunosuppressive tumour microenvironment. Therefore, targeting PTPN2 may provide a means for enhancing the anti-tumour activity of T cells and extending the utility of CAR T cells beyond hematological malignancies to solid cancers.
A recent CRISPR loss-of-function screen in tumour cells identified PTPN2 as a top-hit for the recruitment of T cells and the sensitization of tumours to anti-PD-1 therapy 55,65 . This was reliant on PTPN2-deficiency driving the IFNg-induced and STAT-1-mediated expression of antigen-presentation pathway genes and T cell chemoattractants, such as Cxcl9 in tumour cells 55 . In humans CXCL9 expression is generally associated with increased CD8+ T cell infiltrates and improved overall survival and response to chemotherapy 46 . Our own studies indicate that the deletion of PTPN2 in CAR T cells drives the expression of CXCR3 and the trafficking of CAR T cells to CXCL9/10-expressing mammary tumours. Indeed, we demonstrated that the homing and efficacy of PTPN2-deficient CAR T cells was reliant on tumours expressing STAT-1-driven CXCR3 chemokines. Thus, PTPN2-deficient CAR T cells might be especially effective against tumours such as estrogen receptor negative and triple negative breast cancers 66 or lung cancers 67 that have low PTPN2 levels. However it is important to note that a variety of human tumours, including for example breast, colon and ovarian cancers, can express CXCL9/10/11, or do so after chemotherapy 46,68-

.
Although it may be possible to target PTPN2 systemically with drugs to enhance T cell and CAR T cell responses and potentially achieve synergistic effects through the targeting of PTPN2 in both tumour cells and T cells/CAR T cells, there are several important considerations. First, the currently available PTPN2 inhibitors target the active site of the enzyme and are therefore hydrophilic and have difficulties with bioavailability and pharmacokinetics 74 . Second, we have shown that the inducible deletion of PTPN2 in the hematopoietic compartment of adult non-autoimmune-prone C57BL/6 mice is sufficient to promote the development of systemic inflammation and autoimmunity 8 , whereas PTPN2 deletion in T cells in autoimmune-prone NOD1 mice markedly accelerates type 1 diabetes onset, as well as other autoimmune and inflammatory disorders, including colitis 12 . Therefore, any systemic targeting of PTPN2 might exacerbate immune complications frequently associated with immunotherapy, including cytokine release syndrome that can be life-threatening in CAR T cell therapy 23 . Third we and others have shown that the deletion of PTPN2 in some solid tumours can enhance tumorigenicity 54,66,75 . For example, we have shown that PTPN2 deletion in the liver can facilitate the STAT-3-dependent development of hepatocellular carcinoma in obesity 54 . Accordingly, we propose that the targeting of PTPN2 in adoptively transferred CAR T cells using stabilized siRNAs or CRISPR RNP genome-editing might be a safer and ultimately more effective means for enhancing the clinical efficacy of CAR T cells in solid tumours.
Our studies demonstrate that the deletion of PTPN2 enhances the function of CAR T cells by promoting antigen-induced LCK activation and cytokine-induced STAT-5 signalling. We and others have shown that LCK and STAT-5 can serve as bona fide substrates of PTPN2 in thymocytes/T cells 5,6,9,10,38,39 . In this study we found that correcting the increased LCK or STAT-5 signalling diminished the efficacy of PTPN2-deficient CAR T cells. Our studies indicate that the induction of LCK is necessary for the increased antigen-induced CAR T cell activation, IL-2/IL-15 receptor subunit (CD25, CD122 and CD132) expression and cytotoxicity of PTPN2-deficent CAR T cells, whereas the concomitant direct promotion of STAT5 phosphorylation and cytokine signalling might not only facilitate the acquisition of CTL activity, but also promote homing to CXCL9/10/11 expressing tumours through the induction of CXCR3. IL-2-induced STAT-5 signalling is necessary for the differentiation of activated CD8+ T cells into effectors and the acquisition of CTL activity 49,50 . Although the precise mechanism by which STAT-5 promotes CXCR3 in PTPN2-deficient CAR T cells remains unclear, CXCR3 is elevated in effector CD8+ T cells and previous studies have shown that STAT-5 can drive T-bet and Eomes expression 51 . Consistent with this our studies demonstrate that the elevated STAT-5 signalling in PTPN2deficient CAR T cells was also associated with increased T-bet expression. Importantly, T-bet can drive the expression of CXCR3 52 and both T-bet and Eomes have been shown to be important for the tumour infiltration and anti-tumour activity of cytotoxic CD8+ T cells 53 . Moreover, in the context of viral infection, CXCR3 on CD8+ T cells facilitates T cell homing to infected tissue and the ability of T cells to locate an eliminate infected cells 76 . In our studies we found STAT-5 and the resultant increased CXCR3 promoted the homing of PTPN2-deficient CAR T cells to CXCL9/10expressing mammary tumours within 3 days of adoptive transfer. By contrast significant PTPN2-deficient CAR T cells were not detected in the HER-2+ cerebellum that did not express CXCL9/10. As systemic inflammation, tissue damage, autoimmunity and morbidity were not evident in HER-2 transgenic mice, despite the abundant HER-2 expression in the cerebellum, we conclude that the enhancement of STAT5 signalling limits 'on-target off-tumour' toxicities by promoting the specific homing of CAR T cells to CXCL9/10 expressing tumours.
The results of this study have established the importance of PTPN2 in T cell immunosurveillance and defined a novel target for bolstering the anti-tumour activity of T cells, especially in the context of adoptive T cell therapy. In particular, our findings have defined a novel approach by which to enhance the efficacy of CAR T cells and extend their utility to the treatment of solid tumours without promoting systemic inflammation/autoimmunity and morbidity.

20.
Maude, S.L., et al.     Log-rank (Mantel-Cox) test. In (d) significance was determined using 2-way ANOVA Test. In (e-g) significance was determined using 2-tailed Mann-Whitney U Test.  (a, e) significance was determined using 1-way ANOVA Test. In (b, g, h) significance was determined using 2-way ANOVA Test. In (c) significance was determined using 2-tailed Mann-Whitney U Test. shown. In (a, d, g) significance was determined using 2-tailed Mann-Whitney U Test.

Cell lines and mice
The C57BL/6 mouse breast carcinoma cell line E0771 (a gift from Robin Anderson, Peter MacCallum Cancer Centre) 77 and the C57BL/6 mouse sarcoma cell line 24JK (a gift from Patrick Hwu, NIH, Bethesda, Maryland, USA) 78 were genetically engineered to express truncated human HER-2 (HER-2-E0771) as described previously 79 . The C57BL/6 mouse mammary tumour cell line AT-3 was genetically engineered to express chicken ovalbumin (AT-3-OVA) and has been previously described 80 . The GP+E86 packaging line was generated as previously

Generation of murine CAR T cells.
Splenocytes were isolated from murine spleens by mechanical disruption and contaminating red blood cells were removed by incubation with 1 ml of Blood Cell Lysing Buffer Hybri-Max™ (Sigma-Aldrich) for 7 min at room temperature per spleen.
Lymphocytes (0.5x10 7 /ml) were cultured overnight with anti-CD3e (0.5 µg/ml) and anti-CD28 (0.5 µg/ml) in the presence of 100 IU/ml human IL-2 and 2 ng/ml murine IL-7 in complete T cell medium [RPMI supplemented with 10% FBS, L-glutamine (2mM), penicillin (100 units/mL)/streptomycin (100 μg/mL), non-essential amino acids, Na-pyruvate (1 mM), HEPES (10 mM) and 2-mercaptoethanol (50 μM)]. Dead cells were removed by Ficoll centrifugation (GE Healthcare) according to the manufacturer's instructions. Retrovirus encoding a second generation chimeric antibody receptor (CAR) consisting of an extracellular scFv-anti-human HER-2, a membrane proximal CD8 hinge region and the transmembrane and the cytoplasmic signaling domains of CD28 fused to the cytoplasmic region of CD3ζ (scFv-anti-HER-2-CD28-ζ) was obtained from the supernatant of the GP+E86 packaging line as described previously 35 . Retroviral supernatant was added to non-treated retronectincoated (10 µg/ml) 6-well plates (Takara Bio) and spun for 30 min (1,200 x g) at room temperature. T cells were resuspended in 1 ml of additional retroviral-containing supernatant supplemented with IL-2 and IL-7 and then added to the retronectin-coated plates to a final volume of 5 ml/well with a final T cell concentration of 1 × 10 7 per well. T cells were spun for 90 min (1,200 x g) at room temperature and incubated overnight before the second viral transduction. T cells were maintained in IL-2-and IL-7-containing media and cells used at days 7-8 after transduction.

Generation of human CAR T cells.
Human peripheral blood mononuclear cells (PBMCs) were isolated from normal donor buffy coats. PBMCs were stimulated with anti-human CD3 (OKT3; 30 ng/ml, Ortho Biotech) and human IL-2 (600 IU/ml) in complete T cell medium. Retrovirus  CAR T cells (2x10 5 ) were incubated with HER-2 expressing or HER-2 negative 24JK sarcoma cells (1x10 5 ) in complete T cell medium.

CAR T cells (2x10 5 ) generated from human PBMC were incubated with Lewis
For the assessment of CAR T cell proliferation after anti-CD3e stimulation, purified CD8 + CD44 hi CD62L hi CAR T cells were incubated with plate-bound anti-CD3e for 24 h at 37°C in complete T cell medium. Pre-activated CAR T Cells (2x10 5 ) were labelled with 5 μM CTV and incubated with HER-2 expressing or HER-2 negative 24JK sarcoma cells (1x10 5 ) in complete T cell medium. At various time points, proliferating cells were harvested and CTV dilution was monitored by flow cytometry.

For cell quantification, Nile Red Beads (Prositech) or Flow-Count Fluorospheres
(Beckman Coulter) were added to samples before analysis.

PTPN2-inhibition studies
T cells were incubated rocking on an orbital shaker with PTPN2-inhibitor Compound 8 (100 nM) or vehicle (DMSO 1% v/v) in serum-free RPMI 1640 for 1 h at 37ºC. Cells were washed three times with complete T cell medium and processed for T cell activation studies.

Assessment of autoimmunity in HER-2 TG mice.
Serum liver enzymes aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) were determined with the Transaminase CII kit from Wako Pure Chemical. Serum (20 µl) from HER-2 TG mice or GOT/GPT standards (20 µl) were incubated with GOT or GPT enzyme (500 µl) at 37°C for 5 min. The colour forming solution (500 µl) was added and the reaction terminated with stopping solution (2 ml) after 20 min. Absorbance was read at 555 nM and Karmen units/ml were calculated as sample (OD555 nm)/standard (OD555 nm) * dilution factor * 100 U/ml. Serum anti-nuclear antibodies were detected with the mouse anti-nuclear antibodies Ig's (total IgA+G+M) ELISA Kit from Alpha Diagnostic International.
Serum (1:20 dilution) was incubated on 8-well polystyrene strips coated with ENA (extractable nuclear antigens) at room temperature for 1 h. Plates were washed four times and incubated with Streptavidin-HRP for 30 min followed by five washes.
3,3',5,5'-tetramethylbenzidine was added and reactions developed for 15 min at room temperature in the dark and stopped with 1% sulphuric acid. Absorbance was read at 450 nM and activity units were calculated as sample (OD450 nm)/standard (OD450 nm) * dilution factor * 200 U/ml.

Analysis of tumour-infiltrating T cells.
Tumour bearing mice were sacrificed and tumours were excised and digested at 37°C for 30 min using a cocktail of 1 mg/ml collagenase type IV (Worthington Biochemicals) and 0.02 mg/ml DNase (Sigma-Aldrich) in DMEM supplemented with 2% (v/v) FBS. Cells were passed through a 70 µm cell strainer (BD Biosciences) twice and processed for flow cytometry. For the detection of intracellular cytokines in tumour infiltrating lymphocytes, cells (1x10 6 /200 µl) were stimulated with PMA (20 ng/ml; Sigma-Aldrich) and ionomycin (1 µg/ml; in the presence of GolgiPlug™ and GolgiStop™ (BD Biosciences) for 4 hours at 37°C in complete T cell medium.

24JK sarcoma cells and incubated for four hours at 37ºC in complete T cells medium.
Antigen-specific target cell lysis (24JK-HER-2 cell depletion) was monitored by flow cytometry.

Immunohistochemistry
For brain immunohistochemistry, mice were perfused transcardially with heparinized saline [10,000 units

Quantitative real-time PCR
RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, #15596018) and RNA quality and quantity determined using a NanoDrop 2000 (Thermo Fisher Scientific). mRNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, # 4368814) and processed for quantitative realtime PCR either using the Fast SYBR™ Green Master Mix (Applied Biosystems, # 4385612). Primer sets from PrimePCR™ SYBR® Green Assay (Bio-Rad, #10025636) were utilized to perform quantitative PCR detecting Cxcl9, Cxcl10, Tgfb1, Cd274, H2-K1, and Rps18. Primer sets for HER Nono,and SerpinB14 (ovalbumin) were purchased from Sigma-Aldrich. Relative gene expression (ΔCt) was determined by normalisation to the house-keeping gene Nono throughout the study except for Figure   2C, where Rps18 was used and ΔΔCt analysis performed.

Ptpn2 knock down using siSTABLE™ siRNAs targeting in murine CAR T cells
Ptpn2 was knocked down transiently in HER-2 CAR T cells using nuclease

Overexpression of Ptpn2 in HER-2-E0771 cells
The Tet-on 3G

Statistical analyses
Statistical analyses were performed with Graphpad Prism software 7.0b using the non-parametric using 2-tailed Mann-Whitney U Test, the parametric 2-tailed Student's t test, the 1-way or 2-way ANOVA-test using Turkey or Sidak post-hoc comparison or the Log-rank (Mantel-Cox test) where indicated. p<0.05 was considered as significant.

Animal ethics
All experiments were performed in accordance with the NHMRC Australian Significance in (a, d) was determined using 2-tailed Mann-Whitney U Test.

Supplementary figure 5. PTPN2 deficiency increases T cell infiltration and activation and decreases T cell exhaustion in a syngeneic model of triple negative breast cancer.
AT3-OVA breast cancer cells (1x10 6  Significance in (a-g) was determined using 2-tailed Mann-Whitney U Test. In (h) significance was determined using 2-way ANOVA Test. in the presence of a-CD28 (0.5 µg/ml) and IL-2 (2 ng/ml). After 6 days in culture HER- Representative results (means ± SEM) from at least two independent experiments are shown. In (c, g) significance was determined using 1-way ANOVA Test. In (e) significance was determined using 2-tailed Mann-Whitney U Test.
Cells were stained for CD8 and intracellular IFNg and TNF and the proportion of CD8 + IFNg + and CD8 + IFNg + TNF + CAR T cells determined by flow cytometry. b) Human PBMCs were pretreated with PTPN2-inhibitor (+) or vehicle (-) and stimulated with a-CD3. Cells were stained for CD8, CCR7, CD45RA and CD69 and MFIs for CD69 on CD8 + CCR7 + CD45RA + T cells and CD8 + CCR7 + CD45RA + T cell numbers were determined by flow cytometry. c) HER-2 CAR T cells transfected with GFP