Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103 T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear.

GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells.

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19.

An immunodominant NP-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP-B*07:02-specific CD8 T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52).

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases.

Broad and strong memory CD4 and CD8 T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFNγ based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1.

A Comprehensive Analysis of Key Immune Checkpoint Receptors on Tumor-Infiltrating T Cells From Multiple Types of Cancer.

Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects. This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer.

SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells.

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors.

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.

β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity.

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells.

Within the ruminant system, several possibilities exist to generate dendritic cells migrating out from the tissue into the regional draining lymph nodes as afferent lymph dendritic cells (ALDCs). Here, we analyzed toll-like receptor (TLR) 1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of bovine ALDC. As TLR expression may be influenced by pathogens or vaccines and their adjuvant, it is necessary to understand what TLRs are expressed in a steady-state system to elucidate specific differences and to potentially optimize targeted vaccines.

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity.

Migratory sub-populations of afferent lymphatic dendritic cells differ in their interactions with Mycobacterium bovis Bacille Calmette Guerin.

Understanding how pathogens or vaccine antigens are targeted to dendritic cell (DC) subsets is important for disease pathogenesis studies and vaccine design. We characterised the sub-populations of migrating bovine DC with functional and phenotypic diversity present in pseudoafferent lymph draining the skin. These skin draining DC exist as a series of maturation dependent subsets with differential capacities for antigen uptake and cytokine expression, and include both Langerhans' cells (LC) and dermal derived cells.

Production and characterization of two monoclonal antibodies to bovine tumour necrosis factor alpha (TNF-alpha) and their cross-reactivity with ovine TNF-alpha.

Tumour necrosis factor alpha (TNF-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-alpha.

A toolbox facilitating stable transfection of Eimeria species.

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E.

Development of a simple, sensitive, rapid test which discriminates BCG-vaccinated from Mycobacterium bovis-infected cattle.

Bovine tuberculosis (bTB) is increasing in incidence in the UK. Effective control strategies could involve vaccination; BCG, either alone or in prime-boost strategies, remains the most effective vaccine against bovine tuberculosis. However, BCG vaccination of cattle would require development of diagnostic tests able to accurately discriminate Mycobacterium bovis-infected from BCG-vaccinated animals.

Cross-reactivity of mAbs to human CD antigens with cells from cattle.

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls.

Workshop cluster 1+ gammadelta T-cell receptor T cells from calves express high levels of interferon-gamma in response to stimulation with interleukin-12 and -18.

Gammadelta T-cell receptor(+) T lymphocytes are an important element of the innate immune system. Early production of interferon (IFN)-gamma by gammadelta T cells may have a role in linking innate and adaptive immune responses and contribute to T helper-1 bias. We investigated the role of cytokines in the activation and induction of IFN-gamma secretion by bovine workshop cluster 1(+) (WC1(+)) gammadelta T cells.

Flow cytometric detection of gamma interferon can effectively discriminate Mycobacterium bovis BCG-vaccinated cattle from M. bovis-infected cattle.

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies.

Summary of the animal homologue section of HLDA8.

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish.

The role of dendritic cells in shaping the immune response.

Dendritic cells are central to the initiation of primary immune responses. They are the only antigen-presenting cell capable of stimulating naive T cells, and hence they are pivotal in the generation of adaptive immunity. Dendritic cells also interact with and influence the response of cells of the innate immune system.

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314.

Phenotypic and functional characterization of intraepithelial lymphocytes in a bovine ligated intestinal loop model of enterohaemorrhagic Escherichia coli infection.

Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae. The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood. EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo.