The Parish Laboratory studies the fundamental biology of T cell regulation, with the ultimate goal of discovering new paradigms in T cell biology that could spark the next generation of cancer immunotherapies
Harnessing a patients’ immune system to attack cancer is the most exciting recent breakthrough in cancer treatment. Cancer immunotherapy has raised the possibility of stable disease, or even cure, in a significant portion of patients, but if this dream is to be realised then response rates to therapy must be improved. One of the central mechanisms of action for immunotherapy is reactivation of CD8+ T cells such that they attack the cancer. However, there are still major gaps in our understanding of how to appropriately mobilise these cells against a tumour.
A recurring theme in cancer immunology is that cancers often evade the immune system by hijacking regulatory processes that benefit the host in other contexts. For example, many of the pathways that tumours exploit to avoid immune destruction normally function to prevent autoimmune disease, or limit immunopathology during infection. The Parish laboratory employs a range of non-tumour models, including infection and autoimmune models, to study the broad array of regulatory mechanisms that normally restrain T cell immunity. We use the lessons learned from these systems to design and trial new immunotherapies for cancer.
A current major focus of the laboratory is understanding how different regulatory checkpoints that restrain CD8+ T cells are molecularly controlled, and to what degree these checkpoints are similar or different. Two major checkpoints are known to restrain CD8+ T cells: exhaustion and peripheral tolerance. While these processes were first identified to prevent immunopathology during infection and autoimmunity respectively, there is compelling evidence that both checkpoints are also exploited by cancer. Interestingly, these two regulatory processes operate at fundamentally different points in the immune response: peripheral tolerance prevents responses from being initiated within the tumour draining lymph node, while exhaustion dampens existing responses within the tumour (Fig. 1).
To date the field has focused on how cancer immunotherapy disrupts exhaustion, and how this may contribute to treatment efficacy. In contrast, the contribution of peripheral tolerance to cancer immune evasion, and the clinical potential of disrupting peripheral tolerance for cancer immunotherapy, remains unexplored, in part because the fundamental biology of the tolerance checkpoint is poorly understood. A major research focus of the lab is thus to better define the tolerant state, and characterize to what degree the negative regulatory pathways employed in exhaustion versus tolerance are similar or different. Our initial work has suggested that there are fundamental differences in how exhaustion and tolerance are transcriptionally wired (Wagle et al 2021) (Fig. 2). More broadly, we aim to pioneer cutting-edge technologies technologies (eg. CRISPR engineering (Nüssing et al 2020)) to identify new regulatory pathways that could be targeted for cancer therapy.
Defining the fundamental biology of the tolerance and exhaustion immune checkpoints
The basic biology of the tolerant state remains poorly understood. In particular, it is unclear what pathways control this fate and to what extent these pathways overlap with exhaustion. Comparing exhaustion and tolerance has tremendous potential to both define how these states differ, and identify new regulatory pathways. The pathways that differentially restrain these states are still largely unexplored, providing an exciting and untapped research area for future investigation. By comparing factors induced in both tolerance and exhaustion, we recently identified the transcription factor EGR2 as a novel regulator of exhaustion, although interestingly the regulatory gene programs controlled by EGR2 in tolerance versus exhaustion are distinct (Wagle et al 2021) (Fig. 2). Our current interests are focused on three main areas, and we utilize a combination of autoimmune, infection and cancer models to conduct this research:
- Developing a high-resolution phenotypic description of the tolerant state to better define how it fits into the CD8+ T cell differentiation hierarchy (Parish et al 2009, Van Der Byl et al, in preparation)
- Defining the cellular and molecular signals that enforce tolerance, and determining if they operate similarly during exhaustion (Wagle et al 2021, Wagle et al 2016)
- Identifying new regulatory pathways that control T cell immunity through a range of approaches, including screening strategies (Miosge et al 2017, Wagle et al 2018)
Pioneering new technologies that can provide new insights into fundamental biology remains a major focus of our research. For example, we recently pioneered the first CRISPR-based approach suitable for rapid knock-out cell generation in tolerance models (Nüssing et al 2020), and we have previously used mouse mutagenesis screening to identify novel regulators of T cell immunity (Miosge et al 2017).
Leveraging fundamental biology to design more targeted and effective cancer immunotherapies
Increasing evidence indicate that certain tumours induce tolerance to tumour antigens within the tumour draining lymph node. We are currently defining how peripheral tolerance induced by tumours impairs tumour control by disrupting essential tolerance-associated pathways, and measuring the effect on tumour control in a range of mouse models. Furthermore, given the differences between tolerance and exhaustion, we are determining whether tolerant T cells respond to immunotherapy in a similar manner to exhausted T cells. Our long-term interests involve screening approaches (including drug screens) to identify therapeutic approaches for disrupting tolerance to treat cancer.
More broadly we aim to use the insights gained from our research on fundamental T cell biology, in combination with our newly developed technologies (eg. CRISPR editing), to identify novel strategies to manipulate both T cell exhaustion and/or tolerance in a range of contexts, including CAR T cell therapy. Overall, a major focus of the Parish Laboratory is building and expanding a pipeline spanning fundamental research, through to therapeutic implementation of key concepts in T cell biology for the treatment of cancer.
Determining the association between peripheral tolerance and disease outcome in cancer patients
Despite expanding preclinical evidence that peripheral tolerance restrains anti-tumour immunity, the prevalence and importance of peripheral CD8+ T cell tolerance in people with cancer remains unexplored. Peripheral tolerance has been neglected in patient studies for two main reasons. First, obtaining fresh tissue from patient tumour draining lymph nodes (where cells undergoing tolerance are found) is challenging as lymph nodes are often needed for tumour staging. In contrast, obtaining fresh tumour tissue is much simpler, which has led the field to focus on exhaustion rather than tolerance. Second, identifying cells undergoing tolerance has been challenging due to an absence of definitive markers to identify these cells. We have recently overcome these barriers. Specifically, we have defined reference gene signatures in our preclinical models that enable the differential identification of cells undergoing tolerance versus all other states of differentiation (including exhaustion). We have also established multiple collaborations with clinician researchers at Peter Mac to access fresh tissue from tumour draining lymph nodes across several tumour streams. We are now broadly investigating a range of research questions within these clinical samples including:
- How prevalent is peripheral CD8+ T cell tolerance in the tumour draining lymph node?
- Does elevated tolerance preferentially associate with “cold” tumours that are typically resistant to conventional immunotherapy approaches?
- Are any tumour or lymph node niches preferentially associated with tolerance induction?
- What is the association between elevated tolerance and patient outcome (including responsiveness to therapy)?
More broadly, we are interested in how the immune environment within the tumour draining lymph node may influence the immune environment within the tumour.
1. Wagle MV, Vervoort SJ, Kelly MJ, Van Der Byl W, Peters TJ, Martin BP, Martelotto LG, Nüssing S, Ramsbottom KM, Torpy JR, Knight D, Reading S, Thia K, Miosge LA, Howard DR, Gloury R, Gabriel SS, Utzschneider DT, Oliaro J, Powell JD, Luciani F, Trapani JA, Johnstone RW, Kallies A, Goodnow CC* and Parish IA* (2021). “Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance” Nat. Comm. 12:2782.
In this paper, we identified EGR2 as a novel transcriptional regulator of CD8+ T cell exhaustion in both tumours and chronic infection through direct control of key genes, and indirect control of the exhausted epigenetic state. Strikingly, we found that EGR2, which is a known master regulator of T cell anergy, controls completely different genes in exhaustion. This is the first evidence that exhaustion and anergy are differentially “wired”.
2. Nüssing S, Trapani JA and Parish IA (2020). “Revisiting T cell tolerance as a checkpoint target for cancer immunotherapy” Front. Immunol. 11:589641.
In this review, we argue that inactivation of tumour-specific T cells within the tumour draining lymph node is an immune checkpoint distinct from exhaustion that limits anti-tumour immunity.
3. Nüssing S, House IG, Kearney CJ, Chen AXY, Vervoort SJ, Beavis PA, Oliaro J, Johnstone RW, Trapani JA and Parish IA (2020). “Efficient CRISPR/Cas9 gene editing in uncultured naïve mouse T cells for in vivo studies” J. Immunol. 204:2308-2315.
This methodological study demonstrated that resting, uncultured and quiescent naïve T cells could be edited by CRISPR. The lack of in vitro culture and/or activation makes this editing approach one of the only current methods suitable for studies of early T cell activation (including tolerance models).
4. Wagle MV, Marchingo JM, Howitt J, Tan SS, Goodnow CC* and Parish IA* (2018). “The ubiquitin ligase adaptor NDFIP1 selectively enforces a CD8+ T cell tolerance checkpoint to high dose antigen” Cell Reports 24:577–584.
This study was the first to demonstrate that the negative regulator NDFIP1 enforces CD8+ T cell tolerance, but only in the context of high antigen loads typically associated with T cell anergy.
5. Miosge LA, Sontani Y, Chuah A, Horikawa K, Russell TA, Mei Y, Wagle MV, Howard DR, Enders A, Tscharke DC, Goodnow CC* and Parish IA* (2017). “Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion” Proc. Natl. Acad. Sci. 114:E5216-E5225.
In this paper, we identified a new and (at that time) poorly characterised gene that controls effector T cell expansion after vaccination and infection. Surprisingly, this factor controls expansion via prevention of replication stress selectively within T cells, and appears largely dispensable for protection from replication stress in other cell types. Thus, T cells are uniquely sensitive to replication stress during division.
6. Wagle MV and Parish IA (2016). “FOXO3 is differentially required for CD8+ T cell death during tolerance versus immunity” Immunol. Cell Biol. 94:895-899.
This study demonstrated that the transcriptional pathways responsible for Bim induction and apoptosis are distinct in tolerance versus effector cells.
7. Parish IA*, Marshall HD, Staron MM, Lang PA, Brüstle A, Chen JH, Cui W, Tsui YC, Perry C, Laidlaw BJ, Ohashi PS, Weaver CT and Kaech SM (2014). “Chronic viral infection promotes sustained Th1-derived immunoregulatory IL-10 via BLIMP-1” J. Clin. Invest. 124:3455-3468. * joint corresponding author.
This paper identified that exhausted T cells are a key source of immunosuppressive IL-10 in chronic viral infection, with IL-10 expression induced by the transcription factor BLIMP-1 downstream of chronic TCR signalling. This in turn identified a novel feedback loop in which chronically stimulated, exhausted T cells self-regulate by IL-10 production.
8. Parish IA*, Rao S, Smyth GK, Juelich T, Denyer GS, Davey GM, Strasser A and Heath WR (2009). “The molecular signature of CD8+ T cells undergoing deletional tolerance” Blood 113: 4575-4585. * joint corresponding author.
This study demonstrated that tolerant CD8+ T cells adopt a distinct transcriptional program with both differences and similarities to exhaustion. The gene signatures from this paper have been adopted by the field as hallmark signatures of tolerance.
9. Parish IA, Waithman J, Davey GM, Belz GT, Mintern JD, Kurts C, Sutherland RM, Carbone FR and Heath WR (2009). “Tissue destruction caused by cytotoxic T lymphocytes induces deletional tolerance” Proc. Natl. Acad. Sci. 106: 3901-3906.
This paper demonstrated that in the absence of additional inflammation, antigen release from tissues by CTL killing does not initiate new responses in the lymph node. This has important implications in the context of cancer, as our study challenged the assumption that tumour antigen release by CTL will trigger “epitope spreading”.