Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

ATF4-SLC7A11-GSH axis mediates the acquisition of immunosuppressive properties by activated CD4+ T cells in low arginine condition.

The tumor microenvironment (TME) is restricted in metabolic nutrients including the semi-essential amino acid arginine. While complete arginine deprivation causes T cell dysfunction, it remains unclear how arginine levels fluctuate in the TME to shape T cell fates. Here, we find that the 20-µM low arginine condition, representing the levels found in the plasma of patients with cancers, confers Treg-like immunosuppressive capacities upon activated T cells. In vivo mouse tumor models and human single-cell RNA-sequencing datasets reveal positive correlations between low arginine condition and intratumoral Treg accumulation. Mechanistically, low arginine-activated T cells engage in metabolic and transcriptional reprogramming, using the ATF4-SLC7A11-GSH axis, to preserve their suppressive function. These findings improve our understanding of the role of arginine in human T cell biology with potential applications for immunotherapy strategies.

PemBla: A Phase 1 study of intravesical pembrolizumab in recurrent non-muscle-invasive bladder cancer.

Objectives: This study aimed to investigate the anti-PD-1 inhibitor pembrolizumab as a potential agent for use in non-muscle-invasive bladder cancer (NMIBC) by conducting a Phase 1 safety run-in study to assess the safety and tolerability of intravesical pembrolizumab after transurethral resection of the bladder tumour (TURBT). Patients and methods: Eligible patients had recurrent NMIBC for which adjuvant treatment post TURBT was a reasonable treatment option, Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0-1 and adequate end-organ function. Pembrolizumab was administered by intravesical instillation once weekly for a total of six doses. Intra-patient dose escalation was performed in three paired patient cohorts with doses starting at 50 mg and increasing through 100 mg to a maximum of 200 mg. Adverse events (AEs) were assessed using Common Terminology Criteria for Adverse Events (CTCAE) v4.03 with dose limiting toxicity (DLT) defined as a clinically significant, drug-related, Grade 4 haematological or Grade 3 or higher non-haematological toxicity occurring within 7 days of administration of the first treatment at a given dose for that patient. Results: Six patients were treated with no DLTs seen during dose escalation. Drug-related AEs were of low grade and included dysuria and fatigue. All patients completed six doses of treatment as planned. Pharmacokinetic and pharmacodynamic assays did not detect any pembrolizumab in the serum following repeated intravesical administration, and no changes in peripheral immune cell populations were observed. Conclusions: Administration of intravesical pembrolizumab was well tolerated and did not raise any safety concerns in patients with NMIBC following TURBT. There was no evidence of systemic absorption or systemic immune effects following intravesical administration. Further studies are required to assess whether intravesical administration has anti-tumour activity.

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells.

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.

MAIT cells activate dendritic cells to promote TFH cell differentiation and induce humoral immunity.

Protective immune responses against respiratory pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, are initiated by the mucosal immune system. However, most licensed vaccines are administered parenterally and are largely ineffective at inducing mucosal immunity. The development of safe and effective mucosal vaccines has been hampered by the lack of a suitable mucosal adjuvant. In this study we explore a class of adjuvant that harnesses mucosal-associated invariant T (MAIT) cells. We show evidence that intranasal immunization of MAIT cell agonists co-administered with protein, including the spike receptor binding domain from SARS-CoV-2 virus and hemagglutinin from influenza virus, induce protective humoral immunity and immunoglobulin A production. MAIT cell adjuvant activity is mediated by CD40L-dependent activation of dendritic cells and subsequent priming of T follicular helper cells. In summary, we show that MAIT cells are promising vaccine targets that can be utilized as cellular adjuvants in mucosal vaccines.

Recombinant single-cycle influenza virus with exchangeable pseudotypes allows repeated immunization to augment anti-tumour immunity with immune checkpoint inhibitors.

Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8+ T cell responses. We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.

Decitabine increases neoantigen and cancer testis antigen expression to enhance T-cell-mediated toxicity against glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Despite maximal treatment, median survival remains dismal at 14-24 months. Immunotherapies, such as checkpoint inhibition, have revolutionized management of some cancers but have little benefit for GBM patients. This is, in part, due to the low mutational and neoantigen burden in this immunogenically "cold" tumor. METHODS: U87MG and patient-derived cell lines were treated with 5-aza-2'-deoxycytidine (DAC) and underwent whole-exome and transcriptome sequencing. Cell lines were then subjected to cellular assays with neoantigen and cancer testis antigen (CTA) specific T cells. RESULTS: We demonstrate that DAC increases neoantigen and CTA mRNA expression through DNA hypomethylation. This results in increased neoantigen presentation by MHC class I in tumor cells, leading to increased neoantigen- and CTA-specific T-cell activation and killing of DAC-treated cancer cells. In addition, we show that patients have endogenous cancer-specific T cells in both tumor and blood, which show increased tumor-specific activation in the presence of DAC-treated cells. CONCLUSIONS: Our work shows that DAC increases GBM immunogenicity and consequent susceptibility to T-cell responses in vitro. Our results support a potential use of DAC as a sensitizing agent for immunotherapy.

The P5-type ATPase ATP13A1 modulates major histocompatibility complex I-related protein 1 (MR1)-mediated antigen presentation.

The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included ß2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.

Generation and characterization of HLA-A2 transgenic mice expressing the human TCR 1G4 specific for the HLA-A2 restricted NY-ESO-1157-165 tumor-specific peptide.

BACKGROUND: NY-ESO-1 is a tumor-specific, highly immunogenic, human germ cell antigen of the MAGE-1 family that is a promising vaccine and cell therapy candidate in clinical trial development. The mouse genome does not encode an NY-ESO-1 homolog thereby not subjecting transgenic T-cells to thymic tolerance mechanisms that might impair in-vivo studies. We hypothesized that an NY-ESO-1 T cell receptor (TCR) transgenic mouse would provide the unique opportunity to study avidity of TCR response against NY-ESO-1 for tumor vaccine and cellular therapy development against this clinically relevant and physiological human antigen. METHODS: To study in vitro and in vivo the requirements for shaping an effective T cell response against the clinically relevant NY-ESO-1, we generated a C57BL/6 HLA-A*0201 background TCR transgenic mouse encoding the 1G4 TCR specific for the human HLA-A2 restricted, NY-ESO-1157-165 SLLMWITQC (9C), initially identified in an NY-ESO-1 positive melanoma patient. RESULTS: The HLA-A*0201 restricted TCR was positively selected on both CD4+ and CD8+ cells. Mouse 1G4 T cells were not activated by endogenous autoimmune targets or a large library of non-cognate viral antigens. In contrast, their activation by HLA-A2 NY-ESO-1157-165 complexes was evident by proliferation, CD69 upregulation, interferon-γ production, and interleukin-2 production, and could be tuned using a twofold higher affinity altered peptide ligand, NY-ESO-1157-165V. NY-ESO-1157-165V recombinant vaccination of syngeneic mice adoptively transferred with m1G4 CD8+ T cells controlled tumor growth in vivo. 1G4 transgenic mice suppressed growth of syngeneic methylcholanthrene (MCA) induced HHD tumor cells expressing the full-length human NY-ESO-1 protein but not MCA HHD tumor cells lacking NY-ESO-1. CONCLUSIONS: The 1G4 TCR mouse model for the physiological human TCR against the clinically relevant antigen, NY-ESO-1, is a valuable tool with the potential to accelerate clinical development of NY-ESO-1-targeted T-cell and vaccine therapies.

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

PLGA Nanoparticles Co-encapsulating NY-ESO-1 Peptides and IMM60 Induce Robust CD8 and CD4 T Cell and B Cell Responses.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

HLA-E-restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.

Hepcidin-Mediated Hypoferremia Disrupts Immune Responses to Vaccination and Infection.

BACKGROUND: How specific nutrients influence adaptive immunity is of broad interest. Iron deficiency is the most common micronutrient deficiency worldwide and imparts a significant burden of global disease; however, its effects on immunity remain unclear. METHODS: We used a hepcidin mimetic and several genetic models to examine the effect of low iron availability on T cells in vitro and on immune responses to vaccines and viral infection in mice. We examined humoral immunity in human patients with raised hepcidin and low serum iron caused by mutant TMPRSS6. We tested the effect of iron supplementation on vaccination-induced humoral immunity in piglets, a natural model of iron deficiency. FINDINGS: We show that low serum iron (hypoferremia), caused by increased hepcidin, severely impairs effector and memory responses to immunizations. The intensified metabolism of activated lymphocytes requires the support of enhanced iron acquisition, which is facilitated by IRP1/2 and TFRC. Accordingly, providing extra iron improved the response to vaccination in hypoferremic mice and piglets, while conversely, hypoferremic humans with chronically increased hepcidin have reduced concentrations of antibodies specific for certain pathogens. Imposing hypoferremia blunted the T cell, B cell, and neutralizing antibody responses to influenza virus infection in mice, allowing the virus to persist and exacerbating lung inflammation and morbidity. CONCLUSIONS: Hypoferremia, a well-conserved physiological innate response to infection, can counteract the development of adaptive immunity. This nutrient trade-off is relevant for understanding and improving immune responses to infections and vaccines in the globally common contexts of iron deficiency and inflammatory disorders. FUNDING: Medical Research Council, UK.

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity.

BACKGROUND: Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. METHODS: In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. RESULTS: Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. CONCLUSIONS: Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

The Immune Modulating Properties of Mucosal-Associated Invariant T Cells.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes that express a semi-invariant T cell receptor (TCR) recognizing microbial vitamin B metabolites presented by the highly conserved major histocompatibility complex (MHC) class I like molecule, MR1. The vitamin B metabolites are produced by several commensal and pathogenic bacteria and yeast, but not viruses. Nevertheless, viral infections can trigger MAIT cell activation in a TCR-independent manner, through the release of pro-inflammatory cytokines by antigen-presenting cells (APCs). MAIT cells belong to the innate like T family of cells with a memory phenotype, which allows them to rapidly release Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and in some circumstances Interleukin (IL)-17 and IL-10, exerting an immunomodulatory role on the ensuing immune response, akin to iNKT cells and γδ T cells. Recent studies implicate MAIT cells in a variety of inflammatory, autoimmune diseases, and in cancer. In addition, through the analysis of the transcriptome of MAIT cells activated in different experimental conditions, an important function in tissue repair and control of immune homeostasis has emerged, shared with other innate-like T cells. In this review, we discuss these recent findings, focussing on the understanding of the molecular mechanisms underpinning MAIT cell activation and effector function in health and disease, which ultimately will aid in clinically harnessing this unique, not donor-restricted cell subtype.

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Impacts of combining anti-PD-L1 immunotherapy and radiotherapy on the tumour immune microenvironment in a murine prostate cancer model.

BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.