[Cellular immunotherapy at the University Hospital of Liege : advances, challenges and prospects]. / Immunothérapie cellulaire au CHU de Liège : avancées, défis et perspectives.

Cellular immunotherapy consists in using the cells of the immune system as a therapeutic weapon. In this constantly evolving field, the therapeutic strategies developed at the University Hospital of Liege are hematopoietic stem cell transplantation, mesenchymal stromal cells and targeted therapy with CAR-T cells (Chimeric Antigen Receptor T cells). The first two modalities represent a form of non-targeted cell therapy that has been developed over the past decades. While hematopoietic stem cell transplantation is established as the reference treatment for many hematological diseases, mesenchymal stromal cells are still under investigation in various pathologies (notably Crohn's disease, organ transplantation, COVID-19 and pulmonary fibrosis). By contrast, CAR-T cells represent a recently developed and extremely promising targeted immunotherapy. This therapeutic approach has already revolutionized the treatment of B-cell lymphopathies, and has the potential to do the same for many other diseases in the near future.

L'immunothérapie cellulaire consiste en l'utilisation de cellules du système immunitaire comme arme thérapeutique. Dans ce domaine en évolution constante, les stratégies thérapeutiques développées au CHU de Liège sont la greffe de cellules souches hématopoïétiques, les cellules stromales mésenchymateuses et la thérapie ciblée par cellules CAR-T («Chimeric Antigen Receptor T cells¼). Les deux premières approches représentent une forme de thérapie cellulaire non ciblée, développées depuis de nombreuses années. Si la greffe de cellules souches hématopoïétiques est établie comme le traitement de référence de nombreuses hémopathies, les cellules stromales mésenchymateuses sont, quant à elles, toujours à l'étude dans diverses pathologies (notamment maladie de Crohn, transplantation d'organes, COVID-19 et fibrose pulmonaire). À l'opposé, les cellules CAR-T représentent une immunothérapie ciblée, développée récemment et extrêmement prometteuse. Cette modalité thérapeutique a déjà révolutionné le traitement des lymphopathies B, et elle possède le potentiel d'en faire de même pour de nombreuses autres pathologies dans un avenir proche.

Randomized controlled trial of a group intervention combining self-hypnosis and self-care: secondary results on self-esteem, emotional distress and regulation, and mindfulness in post-treatment cancer patients.

PURPOSE: Cancer patients often report low self-esteem and high emotional distress. Two factors seem particularly linked to these symptoms: emotion regulation strategies and mindfulness. The interest of hypnosis and self-care to relieve these symptoms is not well documented. Our randomized controlled trial aimed at assessing the effect of a group intervention combining self-hypnosis and self-care on self-esteem, emotional distress, emotion regulation, and mindfulness abilities of post-treatment cancer patients, as well as investigating the links between these variables. METHODS: One hundred and four patients who had suffered from cancer were randomized into the intervention group (N = 52) and the wait-list control group (N = 52). They had to answer questionnaires before (T1) and after the intervention (T2). Nine men were excluded from the analyses, leading to a final sample of 95 women with cancer. Group-by-time changes were assessed with MANOVA, and associations with self-esteem and emotional distress were investigated with hierarchical linear regression models. RESULTS: Participants in the intervention group (mean age = 51.65; SD = 12.54) reported better self-esteem, lower emotional distress, a decreased use of maladaptive emotion regulation strategies, and more mindfulness abilities after the intervention, compared to the WLCG. This increase in mindfulness explained 33% of the improvement of self-esteem and 41.6% of the decrease of emotional distress in the intervention group. Self-esteem and emotional distress also predicted each other. CONCLUSION: Our study showed the efficacy of our hypnosis-based intervention to improve all the investigated variables. Mindfulness predicted the improvement of self-esteem and emotional distress. The primary impact of our intervention on mindfulness abilities seems to explain, at least in part, its efficacy. Registration: ClinicalTrials.gov (NCT03144154). Retrospectively registered on the 1st of May, 2017.

Efficacy and safety of aerosolized intra-tracheal dornase alfa administration in patients with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS): a structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may trigger severe pneumonia in coronavirus disease of 2019 (COVID-19) patients through release of damage-associated molecular patterns (DAMPs) and recruitment of neutrophils in the lungs. Activated neutrophils induce inflammation and severe alveolar injury by releasing neutrophil extracellular traps (NETs). The backbones of many DAMPs and NETs are made of extracellular, cell-free DNA decorated with highly toxic compounds such as elastase, myeloperoxidase and citrullinated histones. Dornase alfa is a FDA-approved recombinant human DNAse 1 for the treatment of cystic fibrosis, which cleaves extracellular DNA and may break up cell-free DNA, loosening sticky mucus in the distal airways and reducing NETs-induced toxicity on alveolar pneumocytes. The COVIDornase trial intends to define the impact of aerosolized intra-tracheal dornase alfa administration on the severity and progression of acute respiratory distress syndrome (ARDS) in COVID-19 patients. This drug might make lung mucus thinner and looser, promoting improved clearance of secretions and reduce extracellular double-stranded DNA-induced hyperinflammation in alveoli, preventing further damage to the lungs. TRIAL DESIGN: COVIDornase is a prospective, randomized, controlled, 2-arm (1:1 ratio), multicentric, open-label clinical trial. PARTICIPANTS: The study will recruit mechanically ventilated patients hospitalized in the intensive care unit (ICU) in the recruiting centres (at the time of writing: The Rothschild foundation hospital in Paris, the Strasbourg university hospitals, and Metz-Thionville hospital) who have been diagnosed with COVID-19 and meet ARDS criteria. INCLUSION CRITERIA: - Adult patient (age ≥ 18 years old); - Hospitalized in ICU; - With severe COVID-19 pneumonia and ARDS according to Berlin criteria (PaO2/FiO2 < 300 and PEEP > 5 cmH2O); - Intubated for less than 8 days; - With an anticipated duration of mechanical ventilation > 48 hours; - Carrier of an arterial catheter; - For whom 4 PaO2/FiO2 values over the preceding 24 hours are available; NON-INCLUSION CRITERIA: - Known hypersensitivity to dornase alfa or any of its excipients; - Pregnant or breastfeeding status; - Patient under legal protection. INTERVENTION AND COMPARATOR: Intervention 1, Study group Dornase alfa (Pulmozyme®, Roche, Switzerland) will be administered by aerosol, at a dose of 2500 IU twice daily, 12 hours apart, for 7 consecutive days, using a vibrating mesh nebulizer (Aerogen Solo®, Aerogen, Ireland). The remainder of the management will be performed in accordance with good clinical practice, including mechanical ventilation (protective ventilation, PEEP > 5 cmH2O, tracheal balloon pressure check every 4 hours or automatic device, 30° head of the bed elevation, tidal volume 6-8mL/kg, plateau pressure < 30 cmH2O), neuromuscular blockers if necessary, prone position if PaO2/FiO2 < 150, early enteral nutrition, glycemic control and a sedation protocol based on the RASS score. Intervention 2, Comparator Patients will receive usual care in accordance with good practice (as detailed above), without aerosols. MAIN OUTCOMES: The primary outcome is the occurrence of at least one grade improvement between D0 (inclusion) and D7 in the ARDS scale severity (Berlin criteria). For instance from "severe" to "moderate" or from "moderate" to "mild". RANDOMISATION: All consecutive patients meeting the inclusion criteria will be randomised 1:1 using an eCRF-based, computer-generated randomisation table, either to the dornase alfa arm or to the control arm. An interim analysis will be performed after inclusion of 20 patients. Inclusions may be stopped at the interim analysis per data safety and monitoring board (DSMB) advice, if statistical analyses conclude on the futility or efficacy of the intervention or by other DSMB decision. BLINDING (MASKING): The participants and caregivers will not be blinded to study group assignment. Those assessing the outcomes will be blinded to study group assignment. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): Fifty patients will be randomized to each group, 100 patients in total. TRIAL STATUS: Protocol version number 2, April 29th, 2020. Recruitment is ongoing. The trial started recruitment on the 21st April 2020. We estimate recruitment will finish August 21st 2020. TRIAL REGISTRATION: The trial was registered in ClinicalTrials.gov on 21 April 2020, updated on 8 May 2020. Trial registration number is NCT04355364. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated. This Letter serves as a summary of the key elements of the full protocol.

Efficacy of a hypnosis-based intervention to improve well-being during cancer: a comparison between prostate and breast cancer patients.

BACKGROUND: Prostate and breast cancer can have a lot of negative consequences such as fatigue, sleep difficulties and emotional distress, which decrease quality of life. Group interventions showed benefits to emotional distress and fatigue, but most of these studies focus on breast cancer patients. However, it is important to test if an effective intervention for breast cancer patients could also have benefits for prostate cancer patients. METHODS: Our controlled study aimed to compare the efficacy of a self-hypnosis/self-care group intervention to improve emotional distress, sleep difficulties, fatigue and quality of life of breast and prostate cancer patients. 25 men with prostate cancer and 68 women with breast cancer participated and were evaluated before (T0) and after (T1) the intervention. RESULTS: After the intervention, the breast cancer group showed positive effects for anxiety, depression, fatigue, sleep difficulties, and global health status, whereas there was no effect in the prostate cancer group. We showed that women suffered from higher difficulties prior to the intervention and that their oncological treatments were different in comparison to men. CONCLUSION: The differences in the efficacy of the intervention could be explained by the baseline differences. As men in our sample reported few distress, fatigue or sleep problems, it is likely that they did not improve on these dimensions. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT02569294 and NCT03423927 ). Retrospectively registered in October 2015 and February 2018 respectively.

Review article: mesenchymal stromal cell therapy for inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic relapsing diseases in which pro-inflammatory immune cells and cytokines induce intestinal tissue damage and disability. Mesenchymal stromal cells (MSCs) exert powerful immunomodulatory effects and stimulate tissue repair. AIM: To review the current data on mesenchymal stromal cell therapy in IBD. METHOD: We searched PubMed and 'ClinicalTrials.gov' databases using the terms 'mesenchymal stromal cells', 'mesenchymal stem cell transplantation', 'inflammatory bowel diseases', 'Crohn disease' and 'colitis, ulcerative'. Additional publications were identified from individual article reference lists. RESULTS: MSCs include inhibition of Th1/Th17 lymphocytes and recruitment of regulatory T lymphocytes, induction of antigen-presenting cells into a regulatory-like profile, and stimulation of epithelial cell differentiation and proliferation. More than 200 patients with refractory fistulas have been treated with local injections of MSCs, resulting in complete response in more than half, and in overall response in approximately two thirds of patients. In refractory luminal Crohn's disease, 49 cases of systemic MSC infusions have been reported, while trials with autologous MSCs resulted in mitigated responses, studies using allogeneic MSCs were promising, with around 60% of patients experiencing a response and around 40% achieving clinical remission. CONCLUSIONS: Mesenchymal stromal cells might represent a promising therapy for IBD, especially for Crohn's disease. There remain many unsolved questions concerning the optimal origin and source of mesenchymal stromal cells, dosage and modalities of administration. Moreover, mesenchymal stromal cells still need to prove their effectiveness compared with conventional treatments in randomised controlled trials.

Metastatic signet-ring cell carcinoma of unknown primary origin.

About 3-5% of metastatic cancers originate from an unknown primary origin. Some have a signet-ring cell (SRC) component. We report the medical history of three patients with SRC carcinoma expressing both the oestrogen (ER) and progesterone receptors (PR). Although no primary breast cancer could be identified, we considered these three patients as having metastatic breast cancer. All of them were therefore treated with standard breast anti-hormonal therapies and all demonstrated benefit. The pitfalls of clinical presentation, diagnostic work-up, and treatment are discussed.

[The ideal valvular prosthesis is still to come. Which factors can help decide between mechanical and bioprosthetic heart valve replacement?]. / La prothèse valvulaire idéale n'existe toujours pas. quels facteurs entrent en compte pour orienter les choix d'une valve mécanique ou biologique?

The prevalence of valvular heart diseases reaches 2.5% in the overall population. Aortic valve replacement is one of the most common surgical procedures. We report the story of a female patient whose aortic mechanical valve, implanted at the age of 54 years at the time of a mitral valve repair surgery, had to be replaced 14 years later, due to the development of a subvalvular pannus narrowing the valvular orifice. We use this clinical story to compare the advantages and disadvantages of repair surgery and valve replacement with a biological or mechanical prosthesis, and summarize the latest evidence for the choice of the most adequate prosthesis for a particular patient's profile.

[Mesenchymal stromal cells and organ transplantation]. / Cellules stromales mésenchymateuses et transplantation d'organes.

Mesenchymal stromal cells (MSC) are multipotent and self-renewing cells. MSC are studied for their in vivo and in vitro immunomodulatory effects, in the prevention or the treatment of isehemic injury, and for their potential properties of tissue or organ reconstruction. Over the last few years, the potential role of MSC in organ transplantation has been studied both in vitro and in vivo, and their properties make them an ideal potential cell therapy after solid organ transplantation. A prospective, controlled, phase 1-2 study has been initiated at the CHU of Liege, Belgium. This study assesses the potential risks and benefits of MSC infusion after liver or kidney transplantation. Even if the preliminary results of this study look promising, solely a prospective, randomized, large scale, phase 3 study will allow the clinical confirmation of the theoretical benefits of MSC in solid organ transplantation.

The ETHICA study (part II): simulation study of determinants and variability of ICU physician decisions in patients aged 80 or over.

PURPOSE: To assess physician decisions about ICU admission for life-sustaining treatments (LSTs). METHODS: Observational simulation study of physician decisions for patients aged ≥80 years. Each patient was allocated at random to four physicians who made decisions based on actual bed availability and existence of an additional bed before and after obtaining information on patient preferences. The simulations involved non-invasive ventilation (NIV), invasive mechanical ventilation (IMV), and renal replacement therapy after a period of IMV (RRT after IMV). RESULTS: The physician participation rate was 100/217 (46 %); males without religious beliefs predominated, and median ICU experience was 9 years. Among participants, 85.7, 78, and 62 % felt that NIV, IMV, or RRT (after IMV) was warranted, respectively. By logistic regression analysis, factors associated with admission were age <85 years, self-sufficiency, and bed availability for NIV and IMV. Factors associated with IMV were previous ICU stay (OR 0.29, 95 % CI 0.13-0.65, p = 0.01) and cancer (OR 0.23, 95 % CI 0.10-0.52, p = 0.003), and factors associated with RRT (after IMV) were living spouse (OR 2.03, 95 % CI 1.04-3.97, p = 0.038) and respiratory disease (OR 0.42, 95 % CI 0.23-0.76, p = 0.004). Agreement among physicians was low for all LSTs. Knowledge of patient preferences changed physician decisions for 39.9, 56, and 57 % of patients who disagreed with the initial physician decisions for NIV, IMV, and RRT (after IMV) respectively. An additional bed increased admissions for NIV and IMV by 38.6 and 13.6 %, respectively. CONCLUSIONS: Physician decisions for elderly patients had low agreement and varied greatly with bed availability and knowledge of patient preferences.

In vitro effect of cryptophycin 52 on microtubule assembly and tubulin: molecular modeling of the mechanism of action of a new antimitotic drug.

Cryptophycin 52 (C52) is a new synthetic compound of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The cryptophycin class of compounds acts on microtubules. This report details the mechanism by which C52 substoichiometrically inhibits tubulin self-assembly into microtubules. The inhibition data were analyzed through a model described by Perez-Ramirez [Perez-Ramirez, B., Andreu, J. M., Gorbunoff, M. J., and Timasheff, S. N. (1996) Biochemistry 35, 3277-3285]. We thereby determined the values of the apparent binding constant of the tubulin-C52 complex to the end of a growing microtubule (K(i)) and the apparent binding constant of C52 to tubulin (K(b)). The binding of C52 depended on tubulin concentration, and binding induced changes in the sedimentation pattern of tubulin, which indicates that C52 induces the self-association of tubulin and tubulin aggregates other than microtubules. Using analytical ultracentrifugation and electron microscopy, we show that C52 induces tubulin to form ring-shaped oligomers (single rings). We also show that C52 inhibits the formation of double rings from either GTP- or GDP-tubulin. In addition, the advances made by electron crystallography in understanding the structure of the tubulin and the microtubule allowed us to visualize the putative binding site of C52 and to reconstruct C52-induced ring oligomers by molecular modeling.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Antibodies to C-C chemokine receptor 5 in normal human IgG block infection of macrophages and lymphocytes with primary R5-tropic strains of HIV-1.

In the present study, we demonstrate that normal human IgG for therapeutic use (i.v. Ig) contains natural Abs directed against the CCR5 coreceptor for HIV-1. Abs to CCR5 were isolated from i.v. Ig using an affinity matrix consisting of a synthetic peptide corresponding to the N-terminus of CCR5 coupled to Sepharose. Natural anti-CCR5 Abs inhibited the binding of RANTES to macrophages, demonstrating their interaction with the coreceptor of R5-tropic HIV-1. Affinity-purified anti-CCR5 Ig further inhibited infection of lymphocytes and monocytes/macrophages with primary and laboratory-adapted strains of HIV-1, but did not inhibit infection with X4-tropic HIV. Our results suggest that anti-CCR5 Abs from healthy immunocompetent donors may be suitable for development of novel passive immunotherapy regimens in specific clinical settings in HIV infection.

Homonuclear (1)H-NMR assignment and structural characterization of human immunodeficiency virus type 1 Tat Mal protein.

The transacting transcriptional activator (Tat) is a viral protein essential for activation of the human immunodeficiency virus (HIV) genes, and it plays an important role in HIV induced immunodeficiency. We report the NMR structural characterization of the active Tat Mal variant that belongs to a highly virulent D-subtype HIV type-1 (HIV-1) strain (Mal) found mainly in Africa. A full Tat Mal protein (87 residues) is synthesized. This synthetic protein is active in a transactivation assay with HeLa cells infected with the HIV long terminal repeated noncoding sequences of the HIV-1 provirus (LTR) lac Z gene. Homonuclear (1)H-NMR spectra allows the sequential assignment of the Tat Mal spin systems. Simulating annealing generates 20 conformers with similar folding. The geometry of the mean structure is optimized with energy minimization to obtain a final structure. As the European variant (Tat Bru) the N-terminal region of Tat Mal constitutes the core, and there is a hydrophobic pocket composed of the conserved Trp 11 interacting with several aromatic residues. The two functional regions of Tat (basic and the cysteine-rich regions) are well exposed to the solvent. A short alpha-helix is observed in region V adjacent to the basic region. This alpha helix induces local structural variations compared to the NMR structure of Tat Bru, and it brings the cysteine-rich and basic regions closer. This study suggests that similar folding exists among Tat variants.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative.

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.

CD8 expression allows T cell signaling by monomeric peptide-MHC complexes.

Physiologically, TCR signaling is unlikely to result from the cross-linking of TCR-CD3 complexes, given the low density of specific peptide-MHC complexes on antigen-presenting cells. We therefore have tested directly an alternative model for antigen recognition. We show that monomers of soluble peptide-MHC trigger Ca2+ responses in CD8alphabeta+ T cells. This response is not observed in CD8- T cells and when either the CD8:MHC or CD8:Lck interactions are prevented. This demonstrates that an intact CD8 coreceptor is necessary for effective TCR signaling in response to monomeric peptide-MHC molecules. We propose that this heterodimerization of TCR and CD8 by peptide-MHC corresponds to the physiological event normally involved during antigen-specific signal transduction.

Glimpses at the recognition of peptide/MHC complexes by T-cell antigen receptors.

More than a decade after the first description of the primary structure of a T-cell antigen receptor (TCR), the recent determination of the crystal structure of several unliganded TCR ectodomains and of two TCRs complexed to peptide-MHC ligand provides a structural basis for understanding the initial event that triggers T-cell activation. This review focuses on the topology of the variable (V) domains found in TCRs and immunoglobulins and attempts to delineate the structural features that may render the TCR complementarity-determining regions particularly suited to dock on the peptide/MHC surface. Finally, the available TCR structures provide an opportunity to re-evaluate the molecular basis for intrathymic positive selection as well as the mechanisms that make a given TCR neither infinitely specific, nor so flexible that it engages productively any MHC-binding peptides.

Expression of azurophil and specific granule proteins during differentiation of NB4 cells in neutrophils.

Neutrophils contain several populations of secretory granules with characteristic sets of proteins. Granule proteins are sorted into their respective granule types by temporal regulation of their expression during cell differentiation and/or by specific targeting signals. We investigated the expression of some granule proteins in human promyelocytic NB4 cells. Like other myeloid cell lines which can be differentiated into neutrophils, NB4 cells lack the specific-granule population. We report here that, nevertheless, they express the specific-granule matrix protein lactoferrin, when differentiated with retinoic acid. Lactoferrin and the azurophil-granule protein beta-glucuronidase were simultaneously expressed, whereas myeloperoxidase expression had stopped, showing that azurophil-granule proteins are not all produced concomitantly. Cell fractionation by Percoll gradient revealed that while beta-glucuronidase co-fractionated with myeloperoxidase, lactoferrin was mostly contained in a vesicular compartment free of markers for azurophil granules, plasma membrane, and Golgi. This vesicular compartment was not implicated in regulated exocytosis since it was not mobilized by secretagogues, which, in parallel, induced the release of myeloperoxidase. Furthermore, the specific granule-membrane protein cytochrome b558 also became expressed during NB4-cell differentiation. However, it did not co-localize with lactoferrin but was present in the plasma-membrane fraction. Therefore, differentiation of NB4 cells with retinoic acid leads to the expression of specific- and azurophil-granule proteins and provides a unique cell line model to study the mechanisms involved in the sorting of azurophil- and specific-granule proteins.

No tRNA3Lys unwinding in a complex with HIV NCp7.

The nucleocapsid protein NCp7 of the human immunodeficiency virus (HIV) type 1 is important for the annealing of HIV RNA and tRNA3Lys, the tRNA acting as a primer during reverse transcription of HIV RNA. A wild type NCp7 and a Cys23 mutant having a disrupted zinc finger were analyzed with far UV circular dichroism (CD). CD data analysis revealed that NCp7 has a high content of extended structures in aqueous buffer, decreasing in Cys23 NCp7 and in NCp7 in the absence of zinc. An increase in beta-turn structures is observed in NCp7 bound to tRNA3Lys. Furthermore, CD data shows that Cys23 NCp7 binds tRNA3Lys. The CD spectrum of tRNA3Lys is typical of an A-form helix and retains this structure after binding of NCp7, which demonstrates that NCp7 does not induce tRNA3Lys unwinding. CD spectra of tRNA3Lys were measured from 5 to 80 degrees C to observe CD changes resulting from tRNA3Lys melting. Molecular modeling of the complex identifies two potential tRNA anticodon binding sites in the NCp7 N-terminal region and first zinc finger. In this model, both binding sites can interact with 12 nucleotides in the anticodon domain without requiring a base specificity.