Immune checkpoint therapy responders display early clonal expansion of tumor infiltrating lymphocytes.

Immune checkpoint therapy (ICT) causes durable tumour responses in a subgroup of patients, but it is not well known how T cell receptor beta (TCRβ) repertoire dynamics contribute to the therapeutic response. Using murine models that exclude variation in host genetics, environmental factors and tumour mutation burden, limiting variation between animals to naturally diverse TCRβ repertoires, we applied TCRseq, single cell RNAseq and flow cytometry to study TCRβ repertoire dynamics in ICT responders and non-responders. Increased oligoclonal expansion of TCRβ clonotypes was observed in responding tumours.

CRISPR-Cas9-generated PTCHD1 2489T>G stem cells recapitulate patient phenotype when undergoing neural induction.

An estimated 3.5%-5.9% of the global population live with rare diseases, and approximately 80% of these diseases have a genetic cause.

A surgically optimized intraoperative poly(I:C)-releasing hydrogel prevents cancer recurrence.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects.

Functional validation of variants of unknown significance using CRISPR gene editing and transcriptomics: A Kleefstra syndrome case study.

There are an estimated > 400 million people living with a rare disease globally, with genetic variants the cause of approximately 80% of cases. Next Generation Sequencing (NGS) rapidly identifies genetic variants however they are often of unknown significance. Low throughput functional validation in specialist laboratories is the current ad hoc approach for functional validation of genetic variants, which creating major bottlenecks in patient diagnosis.

CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency.

Background: Over 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays.

Tumour draining lymph node-generated CD8 T cells play a role in controlling lung metastases after a primary tumour is removed but not when adjuvant immunotherapy is used.

Surgical resection of cancer remains the frontline therapy for millions of patients annually, but post-operative recurrence is common, with a relapse rate of around 45% for non-small cell lung cancer. The tumour draining lymph nodes (dLN) are resected at the time of surgery for staging purposes, and this cannot be a null event for patient survival and future response to immune checkpoint blockade treatment. This project investigates cancer surgery, lymphadenectomy, onset of metastatic disease, and response to immunotherapy in a novel model that closely reflects the clinical setting.

Tumor Infiltrating Effector Memory Antigen-Specific CD8 T Cells Predict Response to Immune Checkpoint Therapy.

Immune checkpoint therapy (ICT) results in durable responses in individuals with some cancers, but not all patients respond to treatment. ICT improves CD8 cytotoxic T lymphocyte (CTL) function, but changes in tumor antigen-specific CTLs post-ICT that correlate with successful responses have not been well characterized. Here, we studied murine tumor models with dichotomous responses to ICT.

Pre-treatment tumor neo-antigen responses in draining lymph nodes are infrequent but predict checkpoint blockade therapy outcome.

Immune checkpoint blockade (ICPB) is a powerfully effective cancer therapy in some patients. Tumor neo-antigens are likely main targets for attack but it is not clear which and how many tumor mutations in individual cancers are actually antigenic, with or without ICPB therapy and their role as neo-antigen vaccines or as predictors of ICPB responses. To examine this, we interrogated the immune response to tumor neo-antigens in a murine model in which the tumor is induced by a natural human carcinogen (i.

Impaired T cell proliferation by BET-inhibition impedes adoptive immunotherapy in a murine melanoma model.

Activation of naïve CD8 T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties.

Acquired resistance during adoptive cell therapy by transcriptional silencing of immunogenic antigens.

Immunotherapies such as adoptive cell therapy (ACT) are promising treatments for solid cancers. However, relapsing disease remains a problem and the molecular mechanisms underlying resistance are poorly defined. We postulated that the deregulated epigenetic landscape in cancer cells could underpin the acquisition of resistance to immunotherapy.

Combination immune checkpoint blockade as an effective therapy for mesothelioma.

Mesothelioma is an aggressive asbestos induced cancer with extremely poor prognosis and limited treatment options. Immune checkpoint blockade (ICPB) has demonstrated effective therapy in melanoma and is now being applied to other cancers, including mesothelioma. However, the efficacy of ICPB and which immune checkpoint combinations constitute the best therapeutic option for mesothelioma have yet to be fully elucidated.

Ly49C Impairs NK Cell Memory in Mouse Cytomegalovirus Infection.

NK cells possess inhibitory receptors that are responsible for self-MHC class I recognition; beyond their inhibitory function, accumulating evidence indicates that such receptors confer NK cell functional competence through an unclear process termed "licensing." Ly49C is the main self-specific inhibitory Ly49 receptor in H-2(b) C57BL/6 (B6) mice. We used B6 Ly49C-transgenic and B6 β2 microglobulin (β2m)-knockout Ly49C-transgenic mice to investigate the impact of licensing through this inhibitory receptor in precursor and mature NK cells.

Ly49C-dependent control of MCMV Infection by NK cells is cis-regulated by MHC Class I molecules.

Natural Killer (NK) cells are crucial in early resistance to murine cytomegalovirus (MCMV) infection. In B6 mice, the activating Ly49H receptor recognizes the viral m157 glycoprotein on infected cells. We previously identified a mutant strain (MCMVG1F) whose variant m157 also binds the inhibitory Ly49C receptor.

Targeting of a natural killer cell receptor family by a viral immunoevasin.

Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the 'immunoevasin' m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer.

Functional consequences of natural sequence variation of murine cytomegalovirus m157 for Ly49 receptor specificity and NK cell activation.

The Ly49H activating receptor on C57BL/6 (B6) NK cells plays a key role in early resistance to murine cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. The m157 molecule is also recognized by the Ly49I inhibitory receptor from the 129/J mouse strain. The m157 gene is highly sequence variable among MCMV isolates, with many m157 variants unable to bind Ly49H(B6).

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H.

NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells.

Transcriptional analysis of human cytomegalovirus and rat cytomegalovirus homologues of the M73/M73.5 spliced gene family.

Previous analysis of the M73-to-m74/M75 intergenic region of murine cytomegalovirus (MCMV) identified a family of 3'-co-terminal spliced transcripts that includes M73 and M73.5. The current study investigated whether similar families of spliced genes also exist in the human CMV (HCMV) and rat CMV (RCMV) genomes.

Extensive sequence variation exists among isolates of murine cytomegalovirus within members of the m02 family of genes.

Murine cytomegalovirus (MCMV) is a widely used model for human cytomegalovirus (HCMV) and has facilitated many important discoveries about the biology of CMVs. Most of these studies are conducted using the laboratory MCMV strains Smith and K181. However, wild-derived isolates of MCMV, like HCMV clinical isolates, exhibit genetic variation from laboratory strains, particularly at the ends of their genomes in areas containing known or putative immune-evasion and tropism genes.

NK gene complex haplotype variability and host resistance alleles to murine cytomegalovirus in wild mouse populations.

The NK gene complex (NKC) on mouse chromosome 6 encodes receptors that are expressed on NK cells, such as Ly49H, and is involved in regulating NK cell control of virus infections, such as murine cytomegalovirus (MCMV). In the present study, we investigated the level of allelic heterogeneity in NKC loci in populations of outbred wild mice. This work revealed extensive levels of heterogeneity within two wild mouse populations.

The murine cytomegalovirus M73.5 gene, a member of a 3' co-terminal alternatively spliced gene family, encodes the gp24 virion glycoprotein.

We have identified a novel family of five 3' co-terminal transcripts in murine cytomegalovirus (MCMV) arranged in a tail-to-tail orientation with respect to the MCMV glycoprotein H (gH) gene M75. They share the same exon 2 sequence but possess different exon 1 sequences. Two of these spliced transcripts (M73) encode the MCMV homolog of glycoprotein N (gN) entirely within exon 1.

Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells.

Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation.