Sir Peter MacCallum Department of Oncology - Research Publications

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    A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses
    Todd, KL ; Lai, J ; Sek, K ; Huang, Y-K ; Newman, DM ; Derrick, EB ; Koay, H-F ; Nguyen, D ; Hoang, TX ; Petley, EV ; Chan, CW ; Munoz, I ; House, IG ; Lee, JN ; Kim, JS ; Li, J ; Tong, J ; N. de Menezes, M ; Scheffler, CM ; Yap, KM ; Chen, AXY ; Dunbar, PA ; Haugen, B ; Parish, IA ; Johnstone, RW ; Darcy, PK ; Beavis, PA (NATURE PORTFOLIO, 2023-11-01)
    There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.
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    Single-cell protein expression profiling resolves circulating and resident memory T cell diversity across tissues and infection contexts
    Evrard, M ; Becht, E ; Fonseca, R ; Obers, A ; Park, SL ; Ghabdan-Zanluqui, N ; Schroeder, J ; Christo, SN ; Schienstock, D ; Lai, J ; Burn, TN ; Clatch, A ; House, IG ; Beavis, P ; Kallies, A ; Ginhoux, F ; Mueller, SN ; Gottardo, R ; Newell, EW ; Mackay, LK (CELL PRESS, 2023-07-11)
    Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.
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    CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity
    House, IG ; Derrick, EB ; Sek, K ; Chen, AXY ; Li, J ; Lai, J ; Todd, KL ; Munoz, I ; Michie, J ; Chan, CW ; Huang, Y-K ; Chan, JD ; Petley, E ; Tong, J ; Nguyen, D ; Engel, S ; Savas, P ; Hogg, SJ ; Vervoort, SJ ; Kearney, CJ ; Burr, ML ; Lam, EYN ; Gilan, O ; Bedoui, S ; Johnstone, RW ; Dawson, MA ; Loi, S ; Darcy, PK ; Beavis, PA (CELL PRESS, 2023-08-29)
    CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.
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    CD4(+) CAR T cells persist long-term CD4(+) chimeric antigen receptor T cells in for the long journey
    Chen, AXY ; Derrick, EB ; Beavis, PA ; House, IG (WILEY, 2022-05)
    In a recently published article, Melenhorst et al. performed a longitudinal analysis on chimeric antigen receptor (CAR) T cells isolated from patients over 10 years after therapy, revealing expansion of a long-lived CD4+ CAR T-cell population with a cytotoxic phenotype.
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    TSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models
    Meyran, D ; Zhu, JJ ; Butler, J ; Tantalo, D ; MacDonald, S ; Nguyen, TN ; Wang, M ; Thio, N ; D'Souza, C ; Qin, VM ; Slaney, C ; Harrison, A ; Sek, K ; Petrone, P ; Thia, K ; Giuffrida, L ; Scott, AM ; Terry, RL ; Tran, B ; Desai, J ; Prince, HM ; Harrison, SJ ; Beavis, PA ; Kershaw, MH ; Solomon, B ; Ekert, PG ; Trapani, JA ; Darcy, PK ; Neeson, PJ (AMER ASSOC ADVANCEMENT SCIENCE, 2023-04-05)
    Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.
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    Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses
    Chan, KL ; Gomez, J ; Cardinez, C ; Kumari, N ; Sparbier, CE ; Lam, EYN ; Yeung, MM ; Garciaz, S ; Kuzich, JA ; Ong, DM ; Brown, FC ; Chan, Y-C ; Vassiliadis, D ; Wainwright, EN ; Motazedian, A ; Gillespie, A ; Fennell, KA ; Lai, J ; House, IG ; Macpherson, L ; Ang, C-S ; Dawson, S-J ; Beavis, PA ; Wei, AH ; Burr, ML ; Dawson, MA (CELL PRESS, 2022-10-10)
    There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.
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    IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors
    Giuffrida, L ; Sek, K ; Henderson, MA ; House, IG ; Lai, J ; Chen, AXY ; Todd, KL ; Petley, E ; Mardiana, S ; Todorovski, I ; Gruber, E ; Kelly, MJ ; Solomon, BJ ; Vervoort, SJ ; Johnstone, RW ; Parish, IA ; Neeson, PJ ; Kats, LM ; Darcy, PK ; Beavis, PA (CELL PRESS, 2020-11-04)
    Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.
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    Distinct modulation of IFNγ-induced transcription by BET bromodomain and catalytic P300/CBP inhibition in breast cancer
    Hogg, SJ ; Motorna, O ; Kearney, CJ ; Derrick, EB ; House, IG ; Todorovski, I ; Kelly, MJ ; Zethoven, M ; Bromberg, KD ; Lai, A ; Beavis, PA ; Shortt, J ; Johnstone, RW ; Vervoort, SJ (BMC, 2022-12)
    BACKGROUND: Interferon gamma (IFNγ) is a pro-inflammatory cytokine that directly activates the JAK/STAT pathway. However, the temporal dynamics of chromatin remodeling and transcriptional activation initiated by IFNγ have not been systematically profiled in an unbiased manner. Herein, we integrated transcriptomic and epigenomic profiling to characterize the acute epigenetic changes induced by IFNγ stimulation in a murine breast cancer model. RESULTS: We identified de novo activation of cis-regulatory elements bound by Irf1 that were characterized by increased chromatin accessibility, differential usage of pro-inflammatory enhancers, and downstream recruitment of BET proteins and RNA polymerase II. To functionally validate this hierarchical model of IFNγ-driven transcription, we applied selective antagonists of histone acetyltransferases P300/CBP or acetyl-lysine readers of the BET family. This highlighted that histone acetylation is an antecedent event in IFNγ-driven transcription, whereby targeting of P300/CBP acetyltransferase activity but not BET inhibition could curtail the epigenetic remodeling induced by IFNγ through suppression of Irf1 transactivation. CONCLUSIONS: These data highlight the ability for epigenetic therapies to reprogram pro-inflammatory gene expression, which may have therapeutic implications for anti-tumor immunity and inflammatory diseases.
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    PTP1B Is an Intracellular Checkpoint that Limits T- cell and CAR T- cell Antitumor Immunity
    Wiede, F ; Lu, K-H ; Du, X ; Zeissig, MN ; Xu, R ; Goh, PK ; Xirouchaki, CE ; Hogarth, SJ ; Greatorex, S ; Sek, K ; Daly, RJ ; Beavis, PA ; Darcy, PK ; Tonks, NK ; Tiganis, T (AMER ASSOC CANCER RESEARCH, 2022-03)
    UNLABELLED: Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionized cancer management. To date, these have focused on the blockade of cell-surface checkpoints such as PD-1. Herein we identify protein tyrosine phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that increased PTP1B limits T-cell expansion and cytotoxicity to contribute to tumor growth. T cell-specific PTP1B deletion increased STAT5 signaling, and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacologic inhibition of PTP1B recapitulated the T cell-mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively transferred chimeric antigen receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer. SIGNIFICANCE: Tumors subvert antitumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intratumoral T cells and that its deletion or inhibition enhances T-cell antitumor activity and increases CAR T-cell effectiveness against solid tumors. This article is highlighted in the In This Issue feature, p. 587.
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    Differential location of NKT and MAIT cells within lymphoid tissue
    Johnson, DN ; Ruan, Z ; Petley, E ; Devi, S ; Holz, LE ; Uldrich, AP ; Mak, JYW ; Hor, JL ; Mueller, SN ; McCluskey, J ; Fairlie, DP ; Darcy, PK ; Beavis, PA ; Heath, WR ; Godfrey, D (NATURE PORTFOLIO, 2022-03-08)
    Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αβ T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones, although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone and red pulp of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.