Immune safety challenges facing the preclinical assessment and clinical progression of cell therapies.

The promise of curative outcomes for life-limiting diseases using cell therapies is starting to become a reality, not only for patients with end-stage cancer, but also increasingly for regenerative therapies, including dentistry, ocular, neurodegenerative, and cardiac diseases. The introduction of often genetically modified cells into a patient can come with an extensive range of safety considerations. From an immune perspective, cell-based therapies carry inherent consequences and consideration of factors, such as the cell source (donor-derived autologous cells versus allogeneic cells), the intrinsic cellular nature of the therapy, and engineering/manufacturing methods, all of which influence the likelihood of inducing unwanted immune responses.

Preclinical Evaluation of AZD6422, an Armored Chimeric Antigen Receptor T Cell Targeting CLDN18.2 in Gastric, Pancreatic, and Esophageal Cancers.

Purpose: Claudin 18.2 (CLDN18.2) is a surface membrane protein that is crucial for maintaining tight junctions in gastric mucosal cells and is highly expressed in gastric, esophageal, and pancreatic cancers.

Organoid models: the future companions of personalized drug development.

High failure rates of the current drug development process are driving exemplary changes toward methodologies centered on human diseasemodeling. Organoids are self-organized tissue sub-units resembling their organ of origin and are widely acknowledged for their unique potential in recapitulating human physio-pathological mechanisms. They are transformative for human health by becoming the platform of choice to probe disease mechanisms and advance new therapies.

CD4 T cell memory is impaired by species-specific cytotoxic differentiation, but not by TCF-1 loss.

CD4 T cells are typically considered as 'helper' or 'regulatory' populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4 cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4 CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive.

Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases.

Background: Differences in humoral immunity to coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), between children and adults remain unexplained, and the effect of underlying immune dysfunction or suppression is unknown. Here, we sought to examine the antibody immune competence of children and adolescents with prevalent inflammatory rheumatic diseases, juvenile idiopathic arthritis (JIA), juvenile dermatomyositis (JDM), and juvenile systemic lupus erythematosus (JSLE) against the seasonal human coronavirus (HCoV)-OC43 that frequently infects this age group.

Methods: Sera were collected from JIA (n = 118), JDM (n = 49), and JSLE (n = 30) patients and from healthy control (n = 54) children and adolescents prior to the coronavirus disease 19 (COVID-19) pandemic.

E3 ubiquitin ligase HECTD2 mediates melanoma progression and immune evasion.

The ubiquitin-proteasome system maintains protein homoeostasis, underpins the cell cycle, and is dysregulated in cancer. However, the role of individual E3 ubiquitin ligases, which mediate the final step in ubiquitin-mediated proteolysis, remains incompletely understood. Identified through screening for cancer-specific endogenous retroviral transcripts, we show that the little-studied E3 ubiquitin ligase HECTD2 exerts dominant control of tumour progression in melanoma.

Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein.

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR.

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.

Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents.

Default polyfunctional T helper 1 response to ample signal 1 alone.

CD4 T cells integrate well-defined signals from the T-cell receptor (TCR) (signal 1) and a host of costimulatory molecules (signal 2) to initiate clonal expansion and differentiation into diverse functional T helper (Th) subsets. However, our ability to guide the expansion of context-appropriate Th subsets by deploying these signals in vaccination remains limited. Using cell-based vaccines, we selectively amplified signal 1 by exclusive presentation of an optimized peptide:MHC II (pMHC II) complex in the absence of classic costimulation.

Phosphatase PTPN22 Regulates Dendritic Cell Homeostasis and cDC2 Dependent T Cell Responses.

Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age.

Differential nanoscale organisation of LFA-1 modulates T-cell migration.

Effector T-cells rely on integrins to drive adhesion and migration to facilitate their immune function. The heterodimeric transmembrane integrin LFA-1 (αLβ2 integrin) regulates adhesion and migration of effector T-cells through linkage of the extracellular matrix with the intracellular actin treadmill machinery. Here, we quantified the velocity and direction of F-actin flow in migrating T-cells alongside single-molecule localisation of transmembrane and intracellular LFA-1.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (T) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αβ and γδ CD8 intraepithelial T lymphocytes (IELs) and CD4 T cells.

The protein tyrosine phosphatase PTPN22 negatively regulates presentation of immune complex derived antigens.

A C1858T single nucleotide polymorphism within PTPN22 (which encodes PTPN22) is associated with an enhanced susceptibility to multiple autoimmune diseases including type 1 diabetes and rheumatoid arthritis. Many of the associated autoimmune diseases have an autoantibody component to their pathology. Fc receptors (FcRs) recognise autoantibodies when they bind to autoantigens and form immune complexes.

Protein tyrosine phosphatase PTPN22 regulates LFA-1 dependent Th1 responses.

A missense C1858T single nucleotide polymorphism within PTPN22 is a strong genetic risk factor for the development of multiple autoimmune diseases. PTPN22 encodes a protein tyrosine phosphatase that negatively regulates immuno-receptor proximal Src and Syk family kinases. Notably, PTPN22 negatively regulates kinases downstream of T-cell receptor (TCR) and LFA-1, thereby setting thresholds for T-cell activation.

Protein tyrosine phosphatase PTPN22 is dispensable for dendritic cell antigen processing and promotion of T-cell activation by dendritic cells.

The PTPN22R620W single nucleotide polymorphism increases the risk of developing multiple autoimmune diseases including type 1 diabetes, rheumatoid arthritis and lupus. PTPN22 is highly expressed in antigen presenting cells (APCs) where the expression of the murine disease associated variant orthologue (Ptpn22R619W) is reported to dysregulate pattern recognition receptor signalling in dendritic cells (DCs) and promote T-cell proliferation. Because T-cell activation is dependent on DC antigen uptake, degradation and presentation, we analysed the efficiency of these functions in splenic and GM-CSF bone marrow derived DC from wild type (WT), Ptpn22-/- or Ptpn22R619W mutant mice.

Protein tyrosine phosphatase PTPN22 regulates IL-1β dependent Th17 responses by modulating dectin-1 signaling in mice.

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno-receptors. Fungal β-glucan receptor dectin-1 signals via Syk, and dectin-1 stimulation induces arthritis in mouse models.

Enhancing PET Signal at Target Tissue in Vivo: Dendritic and Multimeric Tris(hydroxypyridinone) Conjugates for Molecular Imaging of αβ Integrin Expression with Gallium-68.

Tris(hydroxypyridinone) chelators conjugated to peptides can rapidly complex the positron-emitting isotope gallium-68 (Ga) under mild conditions, and the resulting radiotracers can delineate peptide receptor expression at sites of diseased tissue in vivo. We have synthesized a dendritic bifunctional chelator containing nine 1,6-dimethyl-3-hydroxypyridin-4-one groups (SCN-HP) that can coordinate up to three Ga ions. This derivative has been conjugated to a trimeric peptide (RGD) containing three peptide groups that target the αβ integrin receptor.

Superresolution imaging of the cytoplasmic phosphatase PTPN22 links integrin-mediated T cell adhesion with autoimmunity.

Integrins are heterodimeric transmembrane proteins that play a fundamental role in the migration of leukocytes to sites of infection or injury. We found that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) inhibits signaling by the integrin lymphocyte function-associated antigen-1 (LFA-1) in effector T cells. PTPN22 colocalized with its substrates at the leading edge of cells migrating on surfaces coated with the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1).

Protein clustering and spatial organization in T-cells.

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

NF-κB signaling mediates homeostatic maturation of new T cells.

Interleukin (IL)-7 is critical for the maintenance of the peripheral T-cell compartment of the adaptive immune system. IL-7 receptor α (IL-7Rα) expression is subject to developmental regulation and new T cells induce expression as they leave the thymus, which is essential for their long-term survival. It is not understood how this expression is regulated.

Primed T cell responses to chemokines are regulated by the immunoglobulin-like molecule CD31.

CD31, an immunoglobulin-like molecule expressed by leukocytes and endothelial cells, is thought to contribute to the physiological regulation T cell homeostasis due to the presence of two immunotyrosine-based inhibitory motifs in its cytoplasmic tail. Indeed, loss of CD31 expression leads to uncontrolled T cell-mediated inflammation in a variety of experimental models of disease and certain CD31 polymorphisms correlate with increased disease severity in human graft-versus-host disease and atherosclerosis. The molecular mechanisms underlying CD31-mediated regulation of T cell responses have not yet been clarified.

Tissue specific deletion of inhibitor of kappa B kinase 2 with OX40-Cre reveals the unanticipated expression from the OX40 locus in skin epidermis.

NF-κB signalling plays an essential role in T cell activation and generation of regulatory and memory populations in vivo. In the present study, we aimed to investigate the role of NF-κB signalling in post-activation T cells using tissue specific ablation of inhibitor of kappa-B kinase 2 expression, an important component of the inhibitor of kappa-B kinase complex in canonical NF-κB signalling. The OX40 antigen is expressed on activated T cells.

Ig gene-like molecule CD31 plays a nonredundant role in the regulation of T-cell immunity and tolerance.

CD31 is an Ig-like molecule expressed by leukocytes and endothelial cells with an established role in the regulation of leukocyte trafficking. Despite genetic deletion of CD31 being associated with exacerbation of T cell-mediated autoimmunity, the contribution of this molecule to T-cell responses is largely unknown. Here we report that tumor and allograft rejection are significantly enhanced in CD31-deficient mice, which are also resistant to tolerance induction.