THEMIS is a priming substrate of non-receptor tyrosine phosphatase PTPN6/SHP1 and plays dual roles during T cell development

THEMIS plays an indispensable role in T cells, but its mechanism of action is highly controversial. Using the systematic proximity labeling methodology PEPSI, we identified THEMIS as an uncharacterized substrate for the phosphatase SHP1. Saturated mutagenesis analysis revealed that THEMIS phosphorylation at the evolutionally conserved Tyr34 residue was oppositely regulated by SHP1 and the kinase LCK. Like THEMIS-/- mice, THEMIS Y34F/Y34F knock-in mice showed a significant decrease in CD4 thymocytes and mature CD4 T cells, but a normal thymic development and peripheral homeostasis of CD8 T cells. Mechanistically, phosphorylated THEMIS induced by TCR activation acts as a "priming substrate" to bind SHP1 and convert its phosphatase activity from basal level to nearly fully activated level, ensuring an appropriate negative regulation of TCR signaling. However, cytokine signaling in CD8 T cells failed to elicit THEMIS Y34 phosphorylation, revealing both phosphorylation-dependent and -independent roles of THEMIS in controlling T cell maturation and expansion.

Computational Construction of a Single-Chain Bi-Paratopic Antibody Allosterically Inhibiting TCR-Staphylococcal Enterotoxin B Binding

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRβ complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRβ chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions.

752 Novel, orally administered HPK1 inhibitors demonstrate anti-tumor efficacy and enhanced immune response

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1) is emerging as a well-renowned, druggable target for T cell-based immunotherapies. HPK1 is a member of the serine/threonine MAP4K family, predominantly expressed in hematopoietic cell lineages and shown to be a negative regulator of the T cell receptor (TCR) signaling pathway. Upon TCR activation, HPK1 is recruited to the proximity of the cell membrane and phosphorylates an adaptor protein SLP-76 at the Ser376 residue which, in turn, abrogates TCR signaling. Other studies point to a potential role of HPK1 in T cell exhaustion as well as in functional re-programming of regulatory T cells. Moreover, mounting evidence suggest that HPK1 kinase activity suppresses the immune functions of a wide range of other immune cell subsets like B cells and dendritic cells. Taken together, these observations support small-molecule HPK1 inhibitors as an attractive modality in cancer immunotherapy either as single agents or in combination with immune checkpoint inhibitors.MethodsActivity of compounds against HPK1 and selected off- and anti-targets was assessed in biochemical assays. Phosphorylation of SLP-76 was measured either by flow cytometry or TR-FRET. Jurkat and primary T cells were activated and cultured in the presence of tested compounds and immunosuppressive agents. Impact on TCR selectivity and T cell function was measured by AlphaLISA and flow cytometry. Target engagement was measured in splenocytes of mice administered orally with tested compounds followed by IP injection of aCD3 antibody. Anti-tumor efficacy of HPK1 inhibitors was assessed in a syngeneic tumor model.ResultsRyvu's proprietary small molecule HPK1 inhibitors exhibit sub-nanomolar activity against human and mouse HPK1 proteins and good selectivity against other TCR pathway kinases. Tested compounds efficiently block phosphorylation of SLP-76 upon TCR engagement. TCR selectivity of Ryvu's inhibitors, measured as a ratio between CD69 and pSer376 SLP-76 inhibition, is on par or superior to reference molecules. Tested compounds are not only able to overcome PGE-2 induced resistance following TCR activation in human PBMCs, inducing elevated IL-2 release but also affect T cell function in co-culture assay. Developed molecules have favorable PK profiles, allowing for sustained target coverage in proposed dosing regimens and demonstrate efficacy in a mammary carcinoma syngeneic model.ConclusionsRyvu has developed potent and selective HPK1 inhibitors with favorable PK and PD profiles, whose activity in vitro translates to in vivo efficacy. Further preclinical work is warranted to select a lead candidate for IND-enabling studies and subsequently clinical studies across a variety of solid tumors.

Spatial heterogeneity of the immune compartment within the lungs of critical COVID-19 patients

Cross AR, Sansom S, Roberts I, Cerundolo L, Melero I, De Andrea C, Landecho MF, Klenerman P,Hester J, Issa F Acute respiratory distress syndrome (ARDS) is a defining feature of severe infection with theSARS-CoV-2 virus. Approaches to understand the immune response during COVID-19 are largelyconfined to characterisation of circulating leukocytes, however this approach excludes the mostrelevant cells that are active at the site of infection and injury. The aim of this study was to characterise the immune landscape across the lungs of COVID-19patients. Lung samples from three critical COVID-19 patients were assessed for histopathology,viral load, and distribution using qPCR, in situ hybridisation and immunohistochemistry.Leukocyte distribution was then assessed, and the transcript profile of selected areas examinedagainst the >1800 genes in the Cancer Transcriptome Atlas panel on the NanoString GeoMxDigital Spatial Profiling platform. Lung samples exhibited a spectrum of typical COVID-19 pathology with diffuse alveolar damageconsistent with hyaline membrane and type II pneumocyte hyperplasia, interstitialinflammation, organising pneumonia and thrombi. All tissues tested positive for SARS-CoV-2RNA using qPCR, whilst spatially resolved techniques revealed only few and sparsely distributedcells carrying the viral nucleocapsid protein. Multiplexed immunofluorescence for lymphocytes(CD3+) and macrophages (CD68+) was used to select areas of immune enrichment for spatialtranscriptomic profiling. These targeted analyses highlighted functional pathways involved inthe interferon gamma response, TCR activation and antigen presentation. Comparison acrossimmune-enriched areas identified a heterogeneity in lung infiltrates with spatial separation ofchemokine and complement production. Our data identify pathological immune pathways thatare amenable to therapeutic intervention in critical disease.

Upregulation of Phosphodiesterase 2A Augments T Cell Activation by Changing cGMP/cAMP Cross-Talk

3′,5′-cyclic adenosine monophosphate (cAMP) is well-known for its diverse immunomodulatory properties, primarily inhibitory effects during T cell activation, proliferation, and production of pro-inflammatory cytokines. A decrease in cAMP levels, due to the hydrolyzing activity of phosphodiesterases (PDE), is favoring inflammatory responses. This can be prevented by selective PDE inhibitors, which makes PDEs important therapeutic targets for autoimmune disorders. In this study, we investigated the specific roles of PDE2A and PDE3B in the regulation of intracellular cAMP levels in different mouse T cell subsets. Unexpectedly, T cell receptor (TCR) activation led to a selective upregulation of PDE2A at the protein level in conventional T cells (Tcon), whereas no changes were detected in regulatory T cells (Treg). In contrast, protein expression of PDE3B was significantly higher in both non-activated and activated Tcon subsets as compared to Treg, with no changes upon TCR engagement. Live-cell imaging of T cells expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor, Epac1-camps, has enabled cAMP measurements in real time and revealed stronger responses to the PDE2A inhibitors in activated vs non-activated Tcon. Importantly, stimulation of intracellular cGMP levels with natriuretic peptides led to an increase of cAMP in non-activated and a decrease of cAMP in activated Tcon, suggesting that TCR activation changes the PDE3B-dependent positive to PDE2A-dependent negative cGMP/cAMP cross-talk. Functionally, this switch induced higher expression of early activation markers CD25 and CD69. This constitutes a potentially interesting feed-forward mechanism during autoimmune and inflammatory responses that may be exploited therapeutically.

Abstract MP229: Signaling Mechanisms Leading To CD4+ T-lymphocyte Activation During Ischemic Heart Failure

CD4 + T-cells turn pathological during chronic heart failure (HF). Phenotypic changes that mediate this transition are unknown. Thus, at 8 weeks post-infarction we conducted limited cell RNA-sequencing on 150 CD4 + T-cells isolated from rodent failing hearts and using ingenuity pathway analysis (z score>2) compared them either with CD4 + T-cells isolated from the mediastinal lymph nodes (mLNs) of the same mice or cardiac CD4 + T-cells from sham mice. T-cells isolated from the mLNs of HF mice showed enhanced TCR signaling (p<2X10 -4 ) with 3-6 fold increase in several downstream mediators such as ITK, MAPK, LCK and Zap70. We also observed heightened leukocyte extravasation signaling (p<2X10 -5 ) with significant increase in PKCθ, and Rho GTPase activating and Ras association domain proteins. These are consistent with enhanced antigen-presentation, and TCR activation during HF. Moreover, we observed significant upregulation of oxidative phosphorylation (p<3X10 -15 ) in cardiac CD4 + T-cells as compared to mLN T-cells with 2-3 fold increase in the gene expression of ATP synthase subunits and cytochrome C indicative of their increased metabolic activity upon infiltration into the failing hearts. Compared to CD4 + T-cells from sham hearts, significant upregulation of inflammatory genes (p<3X10 -5 ), chemotactic factors (p<8X10 -4 ), and several pro-inflammatory cytokine-mediated pathways such as IL-6 (p<2X10 -4 ), IL-1a (p<2X10 -3 ) and TNFα (p<2X10 -3 ) was observed in cardiac CD4 + T-cells from HF mice. Interestingly, we also observed a significant upregulation of homing (p<3X10 -4 ) and connective tissue remodeling genes (p<1.8X10 -3 ) with >8-fold increase in genes like LCN2, SLPI, IGF1, FGFR1, LTF, and TIMP2 required for transmigration and homing of CD4 + T-cells into the ischemic hearts. Our studies show enhanced antigenic CD4 + T-cell activation in the mLNs supporting enhanced pro-inflammatory signaling, metabolic activation, and extravasation into the ischemic hearts.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)–dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non–MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Zap70 Regulates TCR-Mediated Zip6 Activation at the Immunological Synapse

The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4+ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4+ T cells.

Annealing Effect on Structural, Morphological and Electrical Properties by Screen Printed Bunsenite NiO Thick Films.

Thick films of NiO deposited on glass substrate by screen printing technique. The nano powder of AR grade NiO was used for the preparation of thick films. The X-ray diffraction (XRD), Scanning Electron Microscopy and Electrical Characterization was carried out for unannealed and annealed films. The annealed films were at 250 0 C-400 0 C in a muffle furnace. Using characterisation techniques, the success of the synthesised nanoparticles was confirmed. The x-ray diffraction was used for structural characterization which confirms the polycrystalline nature of the films with cubic structure. From the SEM analysis the films show uniformity, roughness, large crystals and agglomeration of particles. The SEM-EDS analysed morphology and chemical compositions. The correlations between structural and morphological properties are reported. The D.C. resistance of the films was measured by half bridge method in air atmosphere at 30OC to 350OC. From the electrical parameters the NiO films shows semiconducting nature. The TCR, activation energy and sheet resistivity, specific surface area were calculated at different annealing temperatures. The electrical conductivity at room temperature was calculated as 4.56 × 𝟏𝟎 −𝟒 (𝜴 ∙ 𝒎) −𝟏