Low-dose decitabine enhances the efficacy of viral cancer vaccines for immunotherapy.

Cancer immunotherapy requires a specific antitumor CD8+ T cell-driven immune response; however, upon genetic and epigenetic alterations of the antigen processing and presenting components, cancer cells escape the CD8+ T cell recognition. As a result, poorly immunogenic tumors are refractory to conventional immunotherapy. In this context, the use of viral cancer vaccines in combination with hypomethylating agents represents a promising strategy to prevent cancer from escaping immune system recognition. In this study, we evaluated the sensitivity of melanoma (B16-expressing ovalbumin) and metastatic triple-negative breast cancer (4T1) cell lines to FDA-approved low-dose decitabine in combination with PeptiCRAd, an adenoviral anticancer vaccine. The two models showed different sensitivity to decitabine in vitro and in vivo when combined with PeptiCRAd. In particular, mice bearing syngeneic 4T1 cancer showed higher tumor growth control when receiving the combinatorial treatment compared to single controls in association with a higher expression of MHC class I on cancer cells and reduction in Tregs within the tumor microenvironment. Furthermore, remodeling of the CD8+ T cell infiltration and cytotoxic activity toward cancer cells confirmed the effect of decitabine in enhancing anticancer vaccines in immunotherapy regimens.

Novel peptide-based oncolytic vaccine for enhancement of adaptive antitumor immune response via co-engagement of innate Fcγ and Fcα receptors.

BACKGROUND: Cancer immunotherapy relies on using the immune system to recognize and eradicate cancer cells. Adaptive immunity, which consists of mainly antigen-specific cytotoxic T cells, plays a pivotal role in controlling cancer progression. However, innate immunity is a necessary component of the cancer immune response to support an immunomodulatory state, enabling T-cell immunosurveillance. METHODS: Here, we elucidated and exploited innate immune cells to sustain the generation of antigen-specific T cells on the use of our cancer vaccine platform. We explored a previously developed oncolytic adenovirus (AdCab) encoding for a PD-L1 (Programmed-Death Ligand 1) checkpoint inhibitor, which consists of a PD-1 (Programmed Cell Death Protein 1) ectodomain fused to an IgG/A cross-hybrid Fc. We coated AdCab with major histocompatibility complex (MHC-I)-restricted tumor peptides, generating a vaccine platform (named PeptiCab); the latter takes advantage of viral immunogenicity, peptide cancer specificity to prime T-cell responses, and antibody-mediated effector functions. RESULTS: As proof of concept, PeptiCab was used in murine models of melanoma and colon cancer, resulting in tumor growth control and generation of systemic T-cell-mediated antitumor responses. In specific, PeptiCab was able to generate antitumor T effector memory cells able to secrete various inflammatory cytokines. Moreover, PeptiCab was able to polarize neutrophils to attain an antigen-presenting phenotype by upregulating MHC-II, CD80 and CD86 resulting in an enhanced T-cell expansion. CONCLUSION: Our data suggest that exploiting innate immunity activates T-cell antitumor responses, enhancing the efficiency of a vaccine platform based on oncolytic adenovirus coated with MHC-I-restricted tumor peptides.

Development of mesothelioma-specific oncolytic immunotherapy enabled by immunopeptidomics of murine and human mesothelioma tumors.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with a poor prognosis. As the available therapeutic options show a lack of efficacy, novel therapeutic strategies are urgently needed. Given its T-cell infiltration, we hypothesized that MPM is a suitable target for therapeutic cancer vaccination. To date, research on mesothelioma has focused on the identification of molecular signatures to better classify and characterize the disease, and little is known about therapeutic targets that engage cytotoxic (CD8+) T cells. In this study we investigate the immunopeptidomic antigen-presented landscape of MPM in both murine (AB12 cell line) and human cell lines (H28, MSTO-211H, H2452, and JL1), as well as in patients' primary tumors. Applying state-of-the-art immuno-affinity purification methodologies, we identify MHC I-restricted peptides presented on the surface of malignant cells. We characterize in vitro the immunogenicity profile of the eluted peptides using T cells from human healthy donors and cancer patients. Furthermore, we use the most promising peptides to formulate an oncolytic virus-based precision immunotherapy (PeptiCRAd) and test its efficacy in a mouse model of mesothelioma in female mice. Overall, we demonstrate that the use of immunopeptidomic analysis in combination with oncolytic immunotherapy represents a feasible and effective strategy to tackle untreatable tumors.

Controlled release of enhanced cross-hybrid IgGA Fc PD-L1 inhibitors using oncolytic adenoviruses.

Immune checkpoint inhibitors have clinical success in prolonging the life of many cancer patients. However, only a minority of patients benefit from such therapy, calling for further improvements. Currently, most PD-L1 checkpoint inhibitors in the clinic do not elicit Fc effector mechanisms that would substantially increase their efficacy. To gain potency and circumvent off-target effects, we previously designed an oncolytic adenovirus (Ad-Cab) expressing an Fc fusion peptide against PD-L1 on a cross-hybrid immunoglobulin GA (IgGA) Fc. Ad-Cab elicited antibody effector mechanisms of IgG1 and IgA, which led to higher tumor killing compared with each isotype alone and with clinically approved PD-L1 checkpoint inhibitors. In this study, we further improved the therapy to increase the IgG1 Fc effector mechanisms of the IgGA Fc fusion peptide (Ad-Cab FT) by adding four somatic mutations that increase natural killer (NK) cell activation. Ad-Cab FT was shown to work better at lower concentrations compared with Ad-Cab in vitro and in vivo and to have better tumor- and myeloid-derived suppressor cell killing, likely because of higher NK cell activation. Additionally, the biodistribution of the Fc fusion peptide demonstrated targeted release in the tumor microenvironment with minimal or no leakage to the peripheral blood and organs in mice. These data demonstrate effective and safe use of Ad-Cab FT, bidding for further clinical investigation.

Peptides-Coated Oncolytic Vaccines for Cancer Personalized Medicine.

Oncolytic Viruses (OVs) work through two main mechanisms of action: the direct lysis of the virus-infected cancer cells and the release of tumor antigens as a result of the viral burst. In this sc.enario, the OVs act as in situ cancer vaccines, since the immunogenicity of the virus is combined with tumor antigens, that direct the specificity of the anti-tumor adaptive immune response. However, this mechanism in some cases fails in eliciting a strong specific T cell response. One way to overcome this problem and enhance the priming efficiency is the production of genetically modified oncolytic viruses encoding one or more tumor antigens. To avoid the long and expensive process related to the engineering of the OVs, we have exploited an approach based on coating OVs (adenovirus and vaccinia virus) with tumor antigens. In this work, oncolytic viruses encoding tumor antigens and tumor antigen decorated adenoviral platform (PeptiCRAd) have been used as cancer vaccines and evaluated both for their prophylactic and therapeutic efficacy. We have first tested the oncolytic vaccines by exploiting the OVA model, moving then to TRP2, a more clinically relevant tumor antigen. Finally, both approaches have been investigated in tumor neo-antigens settings. Interestingly, both genetically modified oncolytic adenovirus and PeptiCRAd elicited T cells-specific anti-tumor responses. However, in vitro cross-representation experiments, showed an advantage of PeptiCRAd as regards the fast presentation of the model epitope SIINFEKL from OVA in an immunogenic rather than tolerogenic fashion. Here two approaches used as cancer oncolytic vaccines have been explored and characterized for their efficacy. Although the generation of specific anti-tumor T cells was elicited in both approaches, PeptiCRAd retains the advantage of being rapidly adaptable by coating the adenovirus with a different set of tumor antigens, which is crucial in personalized cancer vaccines clinical setting.

Regulation of Dynamic Cell Adhesion by Integrin-Integrin Crosstalk.

Most cells express several integrins. The integrins are able to respond to various cellular functions and needs by modifying their own activation state, but in addition by their ability to regulate each other by activation or inhibition. This crosstalk or transdominant regulation is strictly controlled. The mechanisms resulting in integrin crosstalk are incompletely understood, but they often involve intracellular signalling routes also used by other cell surface receptors. Several studies show that the integrin cytoplasmic tails bind to a number of cytoskeletal and adaptor molecules in a regulated manner. Recent work has shown that phosphorylations of integrins and key intracellular molecules are of pivotal importance in integrin-cytoplasmic interactions, and these in turn affect integrin activity and crosstalk. The integrin ß-chains play a central role in regulating crosstalk. In addition to Integrin-integrin crosstalk, crosstalk may also occur between integrins and related receptors, including other adhesion receptors, growth factor and SARS-CoV-2 receptors.

A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines.

Besides the isolation and identification of major histocompatibility complex I-restricted peptides from the surface of cancer cells, one of the challenges is eliciting an effective antitumor CD8+ T-cell-mediated response as part of therapeutic cancer vaccine. Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate >8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-γ enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.

How integrin phosphorylations regulate cell adhesion and signaling.

Cell adhesion is essential for the formation of organs, cellular migration, and interaction with target cells and the extracellular matrix. Integrins are large protein α/ß-chain heterodimers and form a major family of cell adhesion molecules. Recent research has dramatically increased our knowledge of how integrin phosphorylations regulate integrin activity. Phosphorylations determine the signaling complexes formed on the cytoplasmic tails, regulating downstream signaling. α-Chain phosphorylation is necessary for inducing ß-chain phosphorylation in LFA-1, and the crosstalk from one integrin to another activating or inactivating its function is in part mediated by phosphorylation of ß-chains. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus receptor angiotensin-converting enzyme 2 (ACE2) and possible integrin coreceptors may crosstalk and induce a phosphorylation switch and autophagy.

PeptiCHIP: A Microfluidic Platform for Tumor Antigen Landscape Identification.

Identification of HLA class I ligands from the tumor surface (ligandome or immunopeptidome) is essential for designing T-cell mediated cancer therapeutic approaches. However, the sensitivity of the process for isolating MHC-I restricted tumor-specific peptides has been the major limiting factor for reliable tumor antigen characterization, making clear the need for technical improvement. Here, we describe our work from the fabrication and development of a microfluidic-based chip (PeptiCHIP) and its use to identify and characterize tumor-specific ligands on clinically relevant human samples. Specifically, we assessed the potential of immobilizing a pan-HLA antibody on solid surfaces via well-characterized streptavidin-biotin chemistry, overcoming the limitations of the cross-linking chemistry used to prepare the affinity matrix with the desired antibodies in the immunopeptidomics workflow. Furthermore, to address the restrictions related to the handling and the limited availability of tumor samples, we further developed the concept toward the implementation of a microfluidic through-flow system. Thus, the biotinylated pan-HLA antibody was immobilized on streptavidin-functionalized surfaces, and immune-affinity purification (IP) was carried out on customized microfluidic pillar arrays made of thiol-ene polymer. Compared to the standard methods reported in the field, our methodology reduces the amount of antibody and the time required for peptide isolation. In this work, we carefully examined the specificity and robustness of our customized technology for immunopeptidomics workflows. We tested this platform by immunopurifying HLA-I complexes from 1 × 106 cells both in a widely studied B-cell line and in patients-derived ex vivo cell cultures, instead of 5 × 108 cells as required in the current technology. After the final elution in mild acid, HLA-I-presented peptides were identified by tandem mass spectrometry and further investigated by in vitro methods. These results highlight the potential to exploit microfluidics-based strategies in immunopeptidomics platforms and in personalized immunopeptidome analysis from cells isolated from individual tumor biopsies to design tailored cancer therapeutic vaccines. Moreover, the possibility to integrate multiple identical units on a single chip further improves the throughput and multiplexing of these assays with a view to clinical needs.

Novel oncolytic adenovirus expressing enhanced cross-hybrid IgGA Fc PD-L1 inhibitor activates multiple immune effector populations leading to enhanced tumor killing in vitro, in vivo and with patient-derived tumor organoids.

BACKGROUND: Despite the success of immune checkpoint inhibitors against PD-L1 in the clinic, only a fraction of patients benefit from such therapy. A theoretical strategy to increase efficacy would be to arm such antibodies with Fc-mediated effector mechanisms. However, these effector mechanisms are inhibited or reduced due to toxicity issues since PD-L1 is not confined to the tumor and also expressed on healthy cells. To increase efficacy while minimizing toxicity, we designed an oncolytic adenovirus that secretes a cross-hybrid Fc-fusion peptide against PD-L1 able to elicit effector mechanisms of an IgG1 and also IgA1 consequently activating neutrophils, a population neglected by IgG1, in order to combine multiple effector mechanisms. METHODS: The cross-hybrid Fc-fusion peptide comprises of an Fc with the constant domains of an IgA1 and IgG1 which is connected to a PD-1 ectodomain via a GGGS linker and was cloned into an oncolytic adenovirus. We demonstrated that the oncolytic adenovirus was able to secrete the cross-hybrid Fc-fusion peptide able to bind to PD-L1 and activate multiple immune components enhancing tumor cytotoxicity in various cancer cell lines, in vivo and ex vivo renal-cell carcinoma patient-derived organoids. RESULTS: Using various techniques to measure cytotoxicity, the cross-hybrid Fc-fusion peptide expressed by the oncolytic adenovirus was shown to activate Fc-effector mechanisms of an IgA1 (neutrophil activation) as well as of an IgG1 (natural killer and complement activation). The activation of multiple effector mechanism simultaneously led to significantly increased tumor killing compared with FDA-approved PD-L1 checkpoint inhibitor (Atezolizumab), IgG1-PDL1 and IgA-PDL1 in various in vitro cell lines, in vivo models and ex vivo renal cell carcinoma organoids. Moreover, in vivo data demonstrated that Ad-Cab did not require CD8+ T cells, unlike conventional checkpoint inhibitors, since it was able to activate other effector populations. CONCLUSION: Arming PD-L1 checkpoint inhibitors with Fc-effector mechanisms of both an IgA1 and an IgG1 can increase efficacy while maintaining safety by limiting expression to the tumor using oncolytic adenovirus. The increase in tumor killing is mostly attributed to the activation of multiple effector populations rather than activating a single effector population leading to significantly higher tumor killing.

Viral Molecular Mimicry Influences the Antitumor Immune Response in Murine and Human Melanoma.

Molecular mimicry is one of the leading mechanisms by which infectious agents can induce autoimmunity. Whether a similar mechanism triggers an antitumor immune response is unexplored, and the role of antiviral T cells infiltrating the tumor has remained anecdotal. To address these questions, we first developed a bioinformatic tool to identify tumor peptides with high similarity to viral epitopes. Using peptides identified by this tool, we demonstrated that, in mice, preexisting immunity toward specific viral epitopes enhanced the efficacy of cancer immunotherapy via molecular mimicry in different settings. To understand whether this mechanism could partly explain immunotherapy responsiveness in humans, we analyzed a cohort of patients with melanoma undergoing anti-PD1 treatment who had a high IgG titer for cytomegalovirus (CMV). In this cohort of patients, we showed that high levels of CMV-specific antibodies were associated with prolonged progression-free survival and found that, in some cases, peripheral blood mononuclear cells (PBMC) could cross-react with both melanoma and CMV homologous peptides. Finally, T-cell receptor sequencing revealed expansion of the same CD8+ T-cell clones when PBMCs were expanded with tumor or homologous viral peptides. In conclusion, we have demonstrated that preexisting immunity and molecular mimicry could influence the response to immunotherapies. In addition, we have developed a free online tool that can identify tumor antigens and neoantigens highly similar to pathogen antigens to exploit molecular mimicry and cross-reactive T cells in cancer vaccine development.

GAMER-Ad: a novel and rapid method for generating recombinant adenoviruses.

Oncolytic adenoviruses have become ideal agents in the path toward treating cancer. Such viruses have been engineered to conditionally replicate in malignant cells in which certain signaling pathways have been disrupted. Other than such oncolytic properties, the viruses need to activate the immune system in order to sustain a long-term response. Therefore, oncolytic adenoviruses have been genetically modified to express various immune-stimulatory agents to achieve this. However, genetically modifying adenoviruses is very time consuming and labor intensive with the current available methods. In this paper, we describe a novel method we have called GAMER-Ad to genetically modify adenovirus genomes within 2 days. Our method entails the replacement of the gp19k gene in the E3 region with any given gene of interest (GOI) using Gibson Assembly avoiding the homologous recombination between the shuttle and the parental plasmid. In this manuscript as proof of concept we constructed and characterized three oncolytic adenoviruses expressing CXCL9, CXCL10, and interleukin-15 (IL-15). We demonstrate that our novel method is fast, reliable, and simple compared to other methods. We anticipate that our method will be used in the future to genetically engineer oncolytic but also other adenoviruses used for gene therapy as well.

Patient-Derived Organoids for Precision Cancer Immunotherapy.

Cancer immunotherapy has revolutionized the way tumors are treated. Nevertheless, efficient and robust testing platforms are still missing, including clinically relevant human ex vivo tumor assays that allow pretreatment testing of cancer therapies and selection of the most efficient and safe therapy for a specific patient. In the case of immunotherapy, this testing platform would require not only cancer cells, but also the tumor microenvironment, including immune cells. Here, we discuss the applications of patient-derived tumor organoid cultures and the possibilities in using complex immune-organoid cultures to provide preclinical testing platforms for precision cancer immunotherapy.

Bioadhesive supramolecular hydrogel from unprotected, short d,l-peptides with Phe-Phe and Leu-Asp-Val motifs.

The uncapped tripeptide DPhe-Phe-Leu acts as self-assembly template to yield supramolecular hydrogel biomaterials. As an example, self-assembling DPhe-Phe-Leu-Asp-Val contains the LDV bioadhesive motif for ß1 integrin activation. Hydrogels made of the two peptides successfully mimic fibronectin of the extracellular matrix and lead to high cell viability, adhesion, and spreading.

Regulation of cell adhesion: a collaborative effort of integrins, their ligands, cytoplasmic actors, and phosphorylation.

Integrins are large heterodimeric type 1 membrane proteins expressed in all nucleated mammalian cells. Eighteen α-chains and eight ß-chains can combine to form 24 different integrins. They are cell adhesion proteins, which bind to a large variety of cellular and extracellular ligands. Integrins are required for cell migration, hemostasis, translocation of cells out from the blood stream and further movement into tissues, but also for the immune response and tissue morphogenesis. Importantly, integrins are not usually active as such, but need activation to become adhesive. Integrins are activated by outside-in activation through integrin ligand binding, or by inside-out activation through intracellular signaling. An important question is how integrin activity is regulated, and this topic has recently drawn much attention. Changes in integrin affinity for ligand binding are due to allosteric structural alterations, but equally important are avidity changes due to integrin clustering in the plane of the plasma membrane. Recent studies have partially solved how integrin cell surface structures change during activation. The integrin cytoplasmic domains are relatively short, but by interacting with a variety of cytoplasmic proteins in a regulated manner, the integrins acquire a number of properties important not only for cell adhesion and movement, but also for cellular signaling. Recent work has shown that specific integrin phosphorylations play pivotal roles in the regulation of integrin activity. Our purpose in this review is to integrate the present knowledge to enable an understanding of how cell adhesion is dynamically regulated.

A nebulin super-repeat panel reveals stronger actin binding toward the ends of the super-repeat region.

INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super-repeat region into a super-repeat panel, and studied nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.

Phosphorylation of the α-chain in the integrin LFA-1 enables ß2-chain phosphorylation and α-actinin binding required for cell adhesion.

The integrin leukocyte function-associated antigen-1 (LFA-1) plays a pivotal role in leukocyte adhesion and migration, but the mechanism(s) by which this integrin is regulated has remained incompletely understood. LFA-1 integrin activity requires phosphorylation of its ß2-chain and interactions of its cytoplasmic tail with various cellular proteins. The α-chain is constitutively phosphorylated and necessary for cellular adhesion, but how the α-chain regulates adhesion has remained enigmatic. We now show that substitution of the α-chain phosphorylation site (S1140A) in T cells inhibits the phosphorylation of the functionally important Thr-758 in the ß2-chain, binding of α-actinin and 14-3-3 protein, and expression of an integrin-activating epitope after treatment with the stromal cell-derived factor-1α. The presence of this substitution resulted in a loss of cell adhesion and directional cell migration. Moreover, LFA-1 activation through the T-cell receptor in cells expressing the S1140A LFA-1 variant resulted in less Thr-758 phosphorylation, α-actinin and talin binding, and cell adhesion. The finding that the LFA-1 α-chain regulates adhesion through the ß-chain via specific phosphorylation at Ser-1140 in the α-chain has not been previously reported and emphasizes that both chains are involved in the regulation of LFA-1 integrin activity.

HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation.

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

LFA-1 integrin antibodies inhibit leukocyte α4ß1-mediated adhesion by intracellular signaling.

Binding of intercellular adhesion molecule-1 to the ß2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4ß1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4ß1 induce phosphorylation of Thr-758 on the ß2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the ß1-chain of α4ß1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4ß1, inhibit both LFA-1 and α4ß1, inhibit LFA-1 but not α4ß1, or not affect LFA-1 or α4ß1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications.

Specific phosphorylations transmit signals from leukocyte ß2 to ß1 integrins and regulate adhesion.

The regulation of integrins expressed on leukocytes must be controlled precisely, and members of different integrin subfamilies have to act in concert to ensure the proper traffic of immune cells to sites of inflammation. The activation of ß2 family integrins through the T cell receptor or by chemokines leads to the inactivation of very late antigen 4. The mechanism(s) of this cross-talk has not been known. We have now elucidated in detail how the signals are transmitted from leukocyte function-associated antigen 1 and show that, after its activation, the signaling involves specific phosphorylations of ß2 integrin followed by interactions with cytoplasmic signaling proteins. This results in loss of ß1 phosphorylation and a decrease in very late antigen 4 binding to its ligand vascular cell adhesion molecule 1. Our results show how a member of one integrin family regulates the activity of another integrin. This is important for the understanding of integrin-mediated processes.