KRAS mutations are frequent oncogenic drivers in several indications, yet limited targeted therapy options are available. Here we engineered our clinically validated artARENA platform to develop an “off-the-shelf” shared neoantigen vaccine, capable of triggering potent CD8+ T-cell responses against the five most prevalent KRAS mutations (G12D, G12V, G12C, G12R, and G13D). Alternating two-vector therapy (sequential administration of artPICV-based vector followed by artLCMV-based vector, encoding the same optimized antigen construct) induced KRAS neoepitope-specific polyfunctional T-cell responses in HLA transgenic mouse strains, showing direct cytotoxicity against KRAS-mutant cell targets in an in vivo assay. Importantly, no cross-reactivity to wt KRAS was observed, highlighting the safety of the approach. Immunogenicity data in mice was corroborated in vitro using T cell stimulation assays, confirming the antigenicity of the construct. Taken together, these results and the clinically validated favorable safety and immunogenicity profiles of our platform warrant clinical translation of this program with the aim to provide more durable and comprehensive tumor control in patients harboring KRAS mutated tumors.

Humanity has been facing cancer since the beginning of its existence, and the number of new cancer cases grows each year. Immunotherapies based on immune checkpoint inhibition have been intensively developed over the last decade and focus on the blockade of the co-inhibitory molecule complexes such as CTLA-4 with its ligands CD80 and CD86, PD-1 with PD-L1 and PD-L2, and many more. The investigation and development of new immune checkpoints inhibitors is necessary for better cancer treatment. One way of inhibitor evaluation is to assess them in cell-based assays. In this protocol, we focus on describing the cell-based reporter platform for PD-1/PD-L1 inhibitors assessment, which is based on measuring the expression of eGFP under the transcription factor NF-κB, responsible for transcriptional program required for T-cell activation and differentiation.

Innate immune detection of pathogen- and danger-associated molecular patterns (PAMPs/DAMPs) centres on pattern-recognition receptors, with the TLR4/MD-2 complex being uniquely sensitive to trace levels of lipopolysaccharide (LPS) as well as infection-triggered endogenous ligands. While this axis rapidly induces protective cytokine production and upregulation of co-stimulatory molecules, its malfunction can cause pathological hyperinflammation culminating in systemic inflammatory response syndrome (SIRS), highlighting the importance of the development of TLR4 antagonists for the management of immunopathological disorders. Cationic antimicrobial peptides (CAMPs) naturally neutralise LPS by engaging the anionic phosphate groups of lipid A; however, many bacteria evade CAMPs by masking these phosphates with phosphoethanolamine (PE), thereby attenuating electrostatic recognition. In parallel, the PE motif on pathogenic glycans is recognised by the mammalian pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP), which activate complement cascade and play central roles in innate immunity. Building on this paradigm, and analogous to bacterial lipid A remodeling, we synthesised PE-decorated, diglucosamine-based TLR4 antagonists in a highly convergent manner using phosphoramidite and H-phosphonate approaches and evaluated their immunomodulatory activity, biophysical behaviour, and pentraxin recognition. In primary human mononuclear cells, PE-decorated glycolipids attenuated cytokine secretion at micromolar levels, while biophysical analyses showed that they assemble into large, polydisperse aggregates. Zwitterionic glycolipids were recognised and bound by the human pentraxins CRP and SAP, in contrast to their ethanolamine-lacking, negatively charged bis-phosphorylated counterparts. We show that PE modification reprogrammes aggregation behaviour of glycolipids while preserving functional antagonism at TLR4 - albeit with reduced potency - and confers selective recognition by human pentraxins. These results inform the design of next-generation TLR4 antagonists aimed at minimising CAMP sequestration while maintaining efficacy against TLR4-mediated inflammation, with the added potential to engage acute-phase pentraxins.

Antibodies that block PD-1 signaling, known as immune checkpoint inhibitors (ICIs), have revolutionized cancer treatment. Small molecule inhibitors of the PD-1/PD-L1 interaction could serve as promising alternatives to antibody-based ICIs, offering advantages including reduced cost, the option of oral administration, and their small size allows for deeper tissue penetration. Numerous such compounds have been described, but many have not been evaluated in well-defined cellular systems, and comparative studies are scarce. We tested eleven small molecule inhibitors using Jurkat-PD-1-reporter cells activated by stimulator cells expressing PD-L1. Additionally, their effects on T cell reporter activation and viability were evaluated. We found that ARB-272572, INCB086550, Evixapodlin, and PD-1/PD-L1 Inhibitor 3 completely reversed the inhibitory effects of PD-1. Except for PD-1/PD-L1 Inhibitor 3, these compounds blocked PD-1 inhibition with EC50 values in the low nanomolar range. Interestingly, seven PD-1 inhibitors failed to fully block PD-1 inhibition in our cellular assay. BMS-1166 and PD-L1-IN3 showed some blocking capacity but could not fully restore the activation of PD-1 reporter cells. AUNP-12, BMS1, BMS202, CA170, and PD-1/PD-L1-IN-9 were ineffective at reducing PD-1-mediated reporter inhibition. Moreover, our data indicate that adverse effects on T cell reporter activation compromise the activity of several of the tested compounds. In summary, our results revealed that the majority of small molecule PD-1/PD-L1 blockers possess a limited capacity to reverse PD-1 inhibition in a T cell reporter platform and highlight the importance of cellular assays to assess the therapeutic potential of drugs targeting the PD-1/PD-L1 axis.

Occasional complete responses to immune checkpoint inhibitor therapy demonstrate that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) can be immune-sensitive when appropriately targeted. Here, we analyzed AML/MDS patients (n=14) treated with the anti-TIM3 sabatolimab and the hypomethylating agent decitabine in a phase Ib clinical trial (NCT03066648) using single-cell RNA and T cell receptor (TCR) sequencing (n=6) and functional co-culture assays. Unlike T cell-restricted CTLA4 and PD1, TIM3 was broadly expressed across natural killer (NK)-cell, myeloid-cell, and T-cell populations. Therapy induced expansion of cytotoxic NK-cell subsets and enhanced type I interferon signaling. Fewer than 1% of bone marrow CD8+ T cells displayed a canonical exhaustion phenotype, and treatment preferably expanded small CD8+ T-cell clones in responders. Responders exhibited greater expansion of cytotoxic CD4+ T cells and B cells, as exemplified by a patient with pre-existing CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) achieving an outstanding complete response lasting 23 months. Over 20% of this patient's lymphocytes were T-LGLL cells expressing a TCR capable of recognizing autologous blasts. Overall, our results suggest that anti-TIM3 combined with decitabine engages a distinct mechanism of immune activation compared to anti-PD1 and anti-CTLA4, preferentially expanding NK-cell and CD4+ T-cell populations.

Recent clinical trials in AML combining immune checkpoint inhibitors (anti-PD1, anti-CTLA4) with hypomethylating agents have yielded only modest response rates. However, there has been exceptional responders achieving durable complete responses in all these trials. TIM3 is a checkpoint molecule expressed both on immune and leukemic cells, making it an interesting target in AML. Here, we conducted a comprehensive immunomonitoring of a phase Ib trial (NCT03066648) evaluating decitabine in combination with anti-TIM3 antibody sabatolimab (MBG453). We studied paired bone marrow (BM) and peripheral blood samples from 11 unfit newly diagnosed (ND) or relapsed/refractory (R/R) AML patients and 1 MDS patient with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) and flow cytometry. We also performed co-culture assays of primary immune and leukemic cells with scRNA+TCRαβ-seq readout in a patient with a durable complete response. In scRNA+TCRαβ-seq data, HAVCR2 (encoding TIM3) is highly expressed in NK cells, myeloid cells, and unconventional T cells, with some expression in CD8+ T cells. This contrasts with PDCD1 (encoding PD1) and CTLA4, which are primarily expressed in CD8+ and CD4+ T cells, respectively, albeit at low levels in AML. At baseline, responders had higher proportions of CD4+ T cells and B lymphocytes, whereas non-responders had more cytotoxic CD8+ T cells, which was confirmed by flow cytometry. After starting anti-TIM3+HMA therapy, both groups showed increased numbers of CD8+ T cells and NK cells. Responders to anti-TIM3+HMA therapy generally had more mature (CD56dim and adaptive) NK cells, with key transcriptional changes in type I/II interferon (IFN) and NF-κB pathways. Functional co-culture assayswith primary blasts from a complete responder revealed three NK activation states: cell-contact activated, cytokine-secreting, and type I IFN-responsive. NK cells showed enhanced type I IFN responses after therapy correlating with clinical remission, but this response was reduced when NK cells were co-cultured with blasts from a relapse time point, suggesting potential immune evasion. Cytotoxic CD4+ T cells were more abundant and highly clonal in pre-treatment samples from responders. In an exceptional responder with concomitant CD4+ T-LGLL diagnosis, the dominant CD4+ T-LGLL clone comprised 21% of the TCR repertoire at baseline, persisted at remission, but fell to 7% at relapse. Transcriptomic analysis showed upregulation of IFNG and TNF post-therapy, with a subset peaking soon after treatment. To assess whether the T-LGLL clone targets patient's leukemic cells, we engineered Jurkat reporter cells with the TCRαβ from the CD4+ T-LGLL clone and performed co-culture assays with scRNA-seq readout. In comparison to mock transduced cells, T-LGLL reporter cells showed upregulation of type I IFN genes when co-cultured with patient's leukemia cells, especially in presence of antigen-presenting cells. In co-culture of patient's own T-LGLL cells, pre-therapy cells responded strongly to blasts from screening but lost reactivity to relapse blasts, while post-therapy T-LGLL cells retained activity to both. Exhausted CD8+ T cells were rare in AML BM (<0.5% pre-treatment), most CD8+ T cells exhibited effector or memory phenotypes. Functional co-culture assays showed that CD8+ T cells retained robust activation capacity, marked by TNF and IFNG expression, when co-cultured with autologous blasts. Anti-TIM3+HMA therapy preferentially expanded small CD8+ T cell clones in responders, whereas non-responders showed expansion of larger, more cytotoxic clones, some of which were recognized to target viral epitopes. ScRNA-seq analysis of myeloid cells revealed that responders had a higher proportion of differentiated myeloid cells, such as classical monocytes. In the exceptional responder, relapse blasts in co-culture upregulated immune evasion genes (e.g., CD274 encoding PDL1) and showed the highest HLA class I expression, indicating putative immune escape mechanisms from T and NK cells, respectively.Our study provides a comprehensive analysis of anti-TIM3 in combination with decitabine in AML/MDS. We demonstrate that TIM3 blockade modulates the immune landscape by activating mature and adaptive NK cells, promotes cytotoxic CD4+ T cells, and primes small CD8+ T cell clones for expansion. Our results suggest that cytotoxic CD4+ T-LGLL cells may boost responses to immune checkpoint therapy in AML.

Immune checkpoints are critical regulators of immune homeostasis and have become prominent targets in the treatment of various malignancies and autoimmune disorders. While monoclonal antibodies currently dominate checkpoint-targeted therapies, there is a growing interest in alternative approaches, which may offer advantages including smaller size, greater specificity, ease of manufacturing, or a reduced risk of immune-related adverse events. In this study, we focused on the HVEM and its ligand LIGHT, a receptor-ligand pair involved in modulating T cell responses. Overstimulation of HVEM/LIGHT pathway is associated with the pathogenesis of autoimmune diseases and can lead to graft rejection. Therefore, targeting this interaction may offer novel therapeutic strategies for inducing immunosuppression and preventing graft rejection. Utilizing computational approaches, we designed a series of LIGHT-derived peptides. Various simulation techniques, such as molecular dynamics with MMGBSA analyses (with and without NMA), work associated with conformal change and SMD were employed to predict their binding affinities to HVEM. Selected peptides were synthesized and subjected to experimental validation to assess their binding capabilities to HVEM, determined using the spectral shift technique, and inhibitory effects on the HVEM/LIGHT complex formation, evaluated through immunoenzymatic assays and cellular studies. Among these, two peptides demonstrated inhibitory potential, suggesting that they might have utility as scaffold for further optimization. These findings underscore the potential of peptide-based inhibitors in modulating immune checkpoints and pave the way for novel immunotherapeutic strategies.

The virtually monomorphic antigen presentation molecule HLA-E can present self- and non-self peptides to the NKG2A/CD94 co-receptor inhibitory complex expressed on natural killer (NK) cells and to T cell receptors (TCRs) expressed on T cells. HLA-E presents self-peptides to NKG2A/CD94 to regulate tissue homeostasis, whereas HLA-E restricted T cells mediate regulatory and cytotoxic responses toward pathogen-infected cells. In this study, we directly compared HLA-E/peptide recognition and signaling between NKG2A/CD94 and 2 HLA-E restricted TCRs that can recognize self-peptides or identical peptide mimics from the viral UL40 protein of cytomegalovirus using position substituted peptide variants. We show that position 7 is critical for interaction with NKG2A/CD94, whereas position 8 is important for interaction with the TCRs. The Arginine at position 5 of these peptides is an essential residue for recognition by both receptors. Thus, NKG2A/CD94 and TCRs have different requirements for recognition of peptides presented in HLA-E.

Plant-made PD1–Fc fusions engineered for optimized glycosylation and Fc-receptor engagement are highly efficient in blocking PD1/PDL1 interactions and can be cost-effective alternatives to antibody-based immune checkpoint inhibitors. Immune checkpoint inhibitors (ICIs) are antibodies to receptors that have pivotal roles during T-cell activation processes. The programmed cell death 1 (PD1) can be regarded as the primary immune checkpoint and antibodies targeting PD1 or its ligand PDL1 have revolutionized immunotherapy of cancer. However, the majority of patients fail to respond, and treatment resistance as well as immune-related adverse events are commonly associated with this therapy. Alternatives to antibody-based ICIs targeting the PD1 pathway may bear the potential to overcome some of these shortcomings. Here, we have used a plant expression platform based on the tobacco relative Nicotiana benthamiana to generate immunoglobulin fusion proteins harboring the wild type or an affinity-enhanced PD1 ectodomain. We have exploited the versatility of our system to generate variants that differed regarding their glycosylation profile as well as their capability to engage Fc-receptors. Unlike its wild-type counterpart, the affinity-enhanced versions showed strongly augmented capabilities to engage PDL1 in both protein- and cell-based assays. Moreover, in contrast with clinical antibodies, their binding is not affected by the glycosylation status of PDL1. Importantly, we could demonstrate that the plant-made PD1 fusion proteins are highly efficient in blocking inhibitory PD1 signaling in a T cell reporter assay. Taken together, our study highlights the utility of our plant-based protein expression platform to generate biologics with therapeutic potential. Targeting PDL1 with plant derived affinity-enhanced PD1 immunoglobulin fusion proteins may reduce overstimulation associated with antibody-based therapies while retaining favorable features of ICIs such as long serum half-life.

Monocytes and macrophages, as important constituents of the innate immune system, are equipped with multiple Toll-like-receptors (TLRs) to recognize invading pathogens, such as SARS-CoV-2, and mount an antiviral response. Nevertheless, their uncontrolled activation can lead to hyperinflammation seen in severe COVID-19. Surprisingly, we observed that recombinant SARS-CoV-2 Spike (S) and Nucleocapsid (N) proteins triggered only a weak proinflammatory response in human peripheral blood monocytes. By employing THP-1 and Jurkat NF-κB::eGFP reporter cell lines expressing specific TLRs, various TLR ligands and blocking antibodies, we determined that surface TLRs, including TLR2/1, TLR2/6 and TLR4 do not play a major role in SARS-CoV-2 sensing. However, monocytes are potently activated by the replication-competent SARS-CoV-2, and the response correlates with the viral uptake that is observed only in monocytes, but not in lymphocytes. We show that monocyte activation involves two distinct steps. Firstly, SARS-CoV-2 infects monocytes in a process independent of the S protein and the prime SARS-CoV-2 receptor angiotensin-converting enzyme 2. Instead, the alternative SARS-CoV-2 receptor CD147, which is highly expressed on monocytes, recognizes its well-known interaction partners cyclophilins A and B that are incorporated into SARS-CoV-2 virions. Secondly, upon viral uptake via the cyclophilin-CD147 interaction, that can be inhibited by specific CD147 blocking antibodies or competition with recombinant human cyclophilin A and B, SARS-CoV-2 RNA is recognized by TLR7/8 in endosomes, leading to upregulation of tumor necrosis factor (TNF), interleukin (IL)-1β and IL-6, comprising the core hyperinflammatory signature. Taken together, our data reveal a novel mechanism how human monocytes sense SARS-CoV-2 and suggest that targeting the cyclophilin-CD147 axis might be beneficial to alleviate overt myeloid-driven inflammation triggered by SARS-CoV-2 infection.

T cells are important targets for therapeutic intervention in many diseases. Modifying T cell activation via immune checkpoints plays a central role in such approaches. The HVEM/LIGHT complex is one of the stimulatory immune checkpoints involved in T cell activation, and binding of these proteins results in proliferation and development of effector functions of T cells. In patients suffering from autoimmune diseases and in transplant recipients it is desirable to suppress immune responses. This could be achieved by blocking the HVEM and LIGHT interactions. Our studies concern blockage of the formation of the HVEM/LIGHT complex using peptides. To design the inhibitors of this interactions, we relied on the amino acid sequence and the structure of the LIGHT-binding fragments of HVEM. We measured the affinity of designed peptides to LIGHT using SpS technique and tested their ability to inhibit the formation of the HVEM/LIGHT complex using ELISA and cellular studies. That led to the identification of two peptides, namely CRD2e_K54E and CRD2(39-73)e that strongly bind to LIGHT and possess blocking capacities towards HVEM/LIGHT complex formation. Obtained data indicate that HVEM-derived peptides could form the basis for future therapeutics, highlighting the need for further exploration of their immunomodulatory potential.

BTLA and HVEM are key immune checkpoint proteins involved in regulating immune responses. Since HVEM also binds to CD160 at a site that overlaps with the BTLA binding site, CD160-derived fragments were used to design peptide inhibitors targeting this interface. Several peptides were synthesized and assessed for HVEM binding using SpS analysis, and their inhibitory activity was evaluated in ELISA and cell-based assays. One peptide, namely A5, demonstrated strong HVEM binding and effectively blocked the BTLA/HVEM interaction. Molecular docking results revealed that peptide A5 binds to HVEM not only at the BTLA interaction site but also at the region involved in LIGHT binding. Consistent with these findings, ELISA and cell-based assays confirmed that A5 effectively disrupts both BTLA/HVEM and HVEM/LIGHT complex formation. These results suggest that A5 acts as a dual inhibitor of HVEM interactions, with potential therapeutic implications for immune-related disorders.

CD19 chimeric antigen receptor T (CD19CAR-T) cells have achieved promising outcomes in relapsed/refractory B cell malignancies. However, recurrences occur due to the loss of CAR-T cell persistence. We developed dual T/B cell co-stimulatory molecules (CD28 and CD40) in CAR-T cells to enhance intense tumoricidal activity and persistence. CD19.28.40z CAR-T cells promoted pNF-κB and pRelB downstream signaling while diminishing NFAT signaling upon antigen exposure. CD19.28.40z CAR-T cells demonstrated greater proliferation, which translated into effective anti-tumor cytotoxicity in long-term co-culture assay. Repetitive weekly antigen stimulation unveiled continuous CAR-T cell expansion while preserving central memory T cell subset and lower expression of exhaustion phenotypes. The intrinsic genes underlying CD19.28.40z CAR-T cell responses were compared with conventional CARs and demonstrated the up-regulated genes associated with T cell proliferation and memory as well as down-regulated genes related to apoptosis, exhaustion, and glycolysis pathway. Enrichment of genes toward T cell stemness, particularly SELL, IL-7r, TCF7, and KLF2, was observed. Effective and continuing anti-tumor cytotoxicity in vivo was exhibited in both B cell lymphoblastic leukemia and B cell non-Hodgkin lymphoma xenograft models while demonstrating persistent T cell memory signatures. The functional enhancement of CD37.28.40z CAR-T cell activities against CD37+ tumor cells was further validated. The modification of dual T/B cell signaling molecules remarkably maximized the efficacy of CAR-T cell therapy.

T cell co-inhibitory immune checkpoints, such as PD-1 or BTLA, are bona fide targets in cancer therapy. We used a human T cell reporter line to measure transcriptomic changes mediated by PD-1- and BTLA-induced signaling. T cell receptor (TCR)-complex stimulation resulted in the upregulation of a large number of genes but also in repression of a similar number of genes. PD-1 and BTLA signals attenuated transcriptomic changes mediated by TCR-complex signaling: upregulated genes tended to be suppressed and the expression of a significant number of downregulated genes was higher during PD-1 or BTLA signaling. BTLA was a significantly stronger attenuator of TCR-complex-induced transcriptome changes than PD-1. A strong overlap between genes that were regulated indicated quantitative rather than qualitative differences between these receptors. In line with their function as attenuators of TCR-complex-mediated changes, we found strongly regulated genes to be prime targets of PD-1 and BTLA signaling.

The PD-1/PD-L1 complex belongs to the group of inhibitory immune checkpoints and plays a critical role in immune regulation. The PD-1/PD-L1 axis is also responsible for immune evasion of cancer cells, and this complex is one of the main targets of immunotherapies used in oncology. Treatment using immune checkpoint inhibitors is mainly based on antibodies. This approach has great therapeutic potential; however, it also has major drawbacks and can induce immune-related adverse events. Thus, there is a strong need for alternative, non-antibody-based therapies using small molecules, peptides, or peptidomimetics. In the present study, we designed, synthesized, and evaluated a set of PD-1-targeting peptides based on the sequence and structure of PD-L1. The binding of these peptides to PD-1 was investigated using SPR and ELISA. We also assessed their ability to compete with PD-L1 for binding to PD-1 and their inhibitory properties against the PD-1/PD-L1 complex at the cellular level. The best results were obtained for the peptide PD-L1(111–127)(Y112C-I126C), named (L11), which displaced PD-L1 from binding to PD-1 in the competitive assay and inhibited the formation of the PD-1/PD-L1 complex. The (L11) peptide also exhibited strong affinity for PD-1. NMR studies revealed that (L11) does not form a well-defined secondary structure; however, MD simulation indicated that (L11) binds to PD-1 at the same place as PD-L1. After further optimization of the structure, the peptide inhibitor obtained in this study could also be used as a potential therapeutic compound targeting the PD-1/PD-L1 axis.

Tendon diseases pose a significant challenge in regenerative medicine due to the limited healing capacity of this tissue. Successful tendon regeneration requires a combination of angiogenesis, immune response, and tenogenesis processes. An effective tendon engineering (TE) strategy must finely tune this systems’ interplay toward homeostasis.This study explores in vitro the paracrine influence of amniotic epithelial stem cells (AECs) engineered on a validated 3D electrospun PLGA scaffolds on HUVECs (angiogenesis), PBMCs/Jurkat (immune response), and AECs (tenogenic stem cell activation).The results revealed the role of scaffold's topology and topography in significantly modulating the paracrine profile of the cells. In detail, AECs basal release of bioactive molecules was boosted in the cells engineered on 3D scaffolds, in particular VEGF-D, b-FGF, RANTES, and PDGF-BB (p < 0.0001 vs. CMCTR). Moreover, biological tests demonstrated 3D scaffolds' proactive role in potentiating AECs' paracrine inhibition on PBMCs proliferation (CM3D vs. CTR, p < 0.001) and LPS-mediated Jurkat activation with respect to controls (CM3D and CM2D vs. CTR, p < 0.01 and p < 0.05, respectively), without exerting any in vitro pro-angiogenic role in promoting HUVECs proliferation and tubule formation. Teno-inductive paracrine ability of AECs engineered on 3D scaffolds was assessed on co-cultured ones, which formed tendon-like structures. These latter demonstrated an upregulation of tendon-related genes (SCX, THBS4, COL1, and TNMD) and the expression TNMD and COL1 proteins.Overall, this research underscores the pivotal role of the 3D topology and topography of PLGA tendon mimetic scaffolds in orchestrating effective tendon regeneration through modulating cell behavior and crosstalk between engineered stem cells and different subpopulations in the damaged tendon.

Musculoskeletal diseases involving tissue injury comprise tendon, ligament, and muscle injury. Recently, macrophages have been identified as key players in the tendon repair process, but no therapeutic strategy involving dual drug delivery and gene delivery to macrophages has been developed for targeting the two main dysregulated aspects of macrophages in tendinopathy, i.e., inflammation and fibrosis. Herein, the anti-inflammatory and antifibrotic effects of dual-loaded budesonide and serpine1 siRNA lipid-polymer hybrid nanoparticles (LPNs) are evaluated in murine and human macrophage cells. The modulation of the gene and protein expression of factors associated with inflammation and fibrosis in tendinopathy is demonstrated by real time polymerase chain reaction and Western blot. Macrophage polarization to the M2 phenotype and a decrease in the production of pro-inflammatory cytokines are confirmed in macrophage cell lines and primary cells. The increase in the activity of a matrix metalloproteinase involved in tissue remodelling is proven, and studies evaluating the interactions of LPNs with T cells proved that dual-loaded LPNs act specifically on macrophages and do not induce any collateral effects on T cells. Overall, these dual-loaded LPNs are a promising combinatorial therapeutic strategy with immunomodulatory and antifibrotic effects in dysregulated macrophages in the context of tendinopathy.

Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.