Μελέτη των επιπέδων και της λειτουργικότητας των ρυθμιστικών Τ λεμφοκυττάρων σε κακοήθη νοσήματα

Το Πολλαπλούν Μυέλωμα (ΠΜ) είναι μια ανίατη κακοήθεια που χαρακτηρίζεται από τον ανεξέλεγκτο πολλαπλασιασμό παθολογικών πλασματοκυττάρων τα οποία διηθούν το μυελό των οστών και παράγουν περίσσεια μονοκλωνικής ανοσοσφαιρίνης. Τα νέα θεραπευτικά πρωτόκολλα που περιλαμβάνουν, μεταξύ άλλων, τη χρήση αναστολέων του πρωτεασώματος και ανοσοτροποποιητών επιτρέπουν την επίτευξη συμβατικής πλήρους απόκρισης (CR) στην πλειονότητα των ασθενών με ΠΜ. Ωστόσο, συχνά πολλοί ασθενείς υποτροπιάζουν μετά από τη θεραπεία. Για το λόγο αυτό, έχει ενοχοποιηθεί και το μικροπεριβάλλον του μυελού των οστών, το οποίο πιθανά δρα υποστηρικτικά συμβάλλοντας στην κλωνική επέκταση των μυελωματικών πλασματοκυττάρων. Πρόσφατες μελέτες της παθοφυσιολογίας του ΠΜ υποδεικνύουν την πιθανή συμμετοχή δύο ανοσοκατασταλτικών πληθυσμών, των TH17 λεμφοκυττάρων και των κατασταλτικών κυττάρων μυελοειδούς προέλευσης (MDSCs), τα οποία εμφανίζουν αυξημένα ποσοστά στο μυελό των οστών των ασθενών με ΠΜ. Παραδόξως, η μελέτη των ρυθμιστικών Τ λεμφοκυττάρων, τα οποία επίσης χαρακτηρίζονται από ισχυρή ανοσοκατασταλτική δραστικότητα, δεν έχει καταδείξει σαφή συσχέτιση των συγκεκριμένων κυττάρων στην εμφάνιση και πρόοδο της νόσου. Σκοπός της συγκεκριμένης εργασίας ήταν η ανάπτυξη μεθόδου Πολυχρωματικής Κυτταρομετρίας Ροής (ΠΚΡ) για τη μελέτη των επιπέδων και της λειτουργικότητας των Tregs στο μυελό των οστών και στο περιφερικό αίμα ασθενών με ΠΜ στους οποίους, μετά από θεραπεία, ανιχνεύουμε ελάχιστη υπολειπόμενη νόσο (ΕΥΝ+) ή όχι (ΕΥΝ-) στο μυελό των οστών τους. Για τις ανάγκες της συγκεκριμένης εργασίας έγινε φαινοτυπική ανάλυση των Tregs σε δείγματα περιφερικού αίματος και μυελού των οστών από 27 ασθενείς με ΠΜ. Τα δείγματα συλλέχθηκαν από την Αιματολογική-Ογκολογική Κλινική της Ιατρικής Σχολής του ΕΚΠΑ, Γ.Ν.Α. «Αλεξάνδρα». Για την πραγματοποίηση της φαινοτυπικής ανάλυσης των Tregs με ΠΚΡ τα δείγματα και των δύο βιολογικών υλικών σημάνθηκαν με 12 φθορίζοντα αντισώματα χρησιμοποιώντας δύο διαφορετικούς συνδυασμούς οκταχρωμίας: CD3-FITC, CD4-APC/Cy7, CD25-APC, FoxP3-PE, CD8-PerCP/Cy5-5, Ki67-AmCyan, CD45RA-PE/Cy7, CD39-Pacific Blue, CD45RO-PerCP/Cy5-5, CTLA-4-Pacific Blue, CD127-AmCyan, HLADR-PE/Cy7. Παράλληλα με τη φαινοτυπική ανάλυση των Tregs, στα δείγματα μυελού των οστών και περιφερικού αίματος νεοδιαγνωσθέντων ασθενών έγινε ταυτοποίηση του υποτύπου της παραπρωτεΐνης, όπου αυτό ήταν εφικτό, ενώ οι ασθενείς που βρίσκονταν σε πλήρη κλινική ύφεση μετά από θεραπεία, κατηγοριοποιήθηκαν ανάλογα με την εμφάνιση ή όχι ΕΥΝ στα δείγματα μυελού των οστών με τη χρήση ΠΚΡ. Με βάση τα παραπάνω έγινε (%) ποσοτικός προσδιορισμός των επιπέδων των Tregs και μελέτη της λειτουργικότητάς τους ξεχωριστά σε τρεις ομάδες ασθενών: α) νεοδιαγνωσθέντες ασθενείς, β) ΕΥΝ+ ασθενείς και γ) ΕΥΝ- ασθενείς. Σύμφωνα με τα αποτελέσματά μας, τα Tregs υπερέχουν αριθμητικά στο περιφερικό αίμα συγκριτικά με το μυελό των οστών και στις τρεις ομάδες ασθενών (νεοδιαγνωσθέντες, ΕΥΝ+, ΕΥΝ-) που εξετάσαμε, ενώ οι EYN- ασθενείς εμφάνισαν τα υψηλότερα ποσοστά CD3+CD4+CD25hiFoxP3+ Tregs και CD3+CD4+CD25hiFoxP3+CD127lo Tregs τόσο στο σύνολο των CD4+ T λεμφοκυττάρων όσο και στο σύνολο των εμπύρηνων κυττάρων για κάθε αναλυθέν δείγμα. Παράλληλα, οι νεοδιαγνωσθέντες ασθενείς χαρακτηρίζονταν από υψηλότερα ποσοστά Tregs επί των CD4+ T λεμφοκυττάρων στο περιφερικό αίμα σε σχέση με το μυελό των οστών, ενώ στους ΕΥΝ+ και ΕΥΝ- παρατηρήθηκε η αντίστροφη εικόνα. Όσον αφορά τη μελέτη του λειτουργικού προφίλ των CD3+CD4+CD25hiFoxP3+ Tregs, δεν παρατηρήθηκαν διαφορές στην έκφραση των αντιγονικών δεικτών που μελετήσαμε μεταξύ μυελού των οστών και περιφερικού αίματος ανά ομάδα ασθενών, ωστόσο οι EYN+ ασθενείς εμφάνισαν υψηλότερα ποσοστά CD39+ Tregs. Τα παραπάνω αποτελέσματα αποτελούν μια πρώτη ένδειξη ότι τα Tregs των EYN+ ασθενών πιθανά εμφανίζουν ισχυρότερα ανοσοκατασταλτικό φαινότυπο, διευκολύνοντας την ανοσοδιαφυγή των μυελωματικών κυττάρων, παρά το γεγονός ότι σε απόλυτους αριθμούς δεν παρουσιάζουν αριθμητική αύξηση. Ακόμη, είναι πιθανό οι φαρμακευτικοί παράγοντες που χρησιμοποιούνται στα θεραπευτικά πρωτόκολλα να μεταβάλλουν άμεσα ή έμμεσα το πρότυπο μετανάστευσης και αγκυροβόλησης των Tregs από το περιφερικό αίμα προς το μυελό των οστών ή αντίστροφα. Παρόλα αυτά, απαιτείται ανάλυση περισσότερων δειγμάτων για την αποσαφήνιση του ρόλου των Tregs στην παθοφυσιολογία του ΠΜ.Multiple Myeloma (MM) is an incurable malignancy characterized by the uncontrolled proliferation of aberrant plasma cells that infiltrate the bone marrow and overproduce abnormal immunoglobulin fragments. Modern therapeutic regimens that include proteasome inhibitors and immunomodulators allow for the achievement of a complete response (CR) in the majority of MM patients. Nevertheless, many patients often relapse after treatment. For this reason, the bone marrow microenvironment has also been implicated, due to its’ potential supportive role in contributing to the clonal expansion of myeloma plasma cells. Recent studies on the pathophysiology of MM indicate a possible involvement of two immunosuppressive populations, TH17 lymphocytes and Myeloid Derived Suppressor Cells (MDSCs), which appear to be increased in the bone marrow of MM patients. Surprisingly, studies performed on regulatory T cells (Tregs), which exhibit potent immunosuppressive activity, have not yet demonstrated the correlation of these cells with disease onset and progression. The purpose of this project was to develop a method based on Multiparameter Flow Cytometry (MFC) to study the levels and functionality of Tregs in the bone marrow and peripheral blood of MM patients, including those with detectable levels of Minimal Residual Disease (MRD+) or not (MRD-) in their bone marrow. For this purpose, we performed immunophenotypic analysis of Tregs in peripheral blood and bone marrow samples from 27 MM patients. The samples were collected at the Department of Clinical Therapeutics, School of Medicine, University of Athens. In order to identify Tregs and study their functional profile, samples of both biologic materials were labeled with 12 fluorescent antibodies comprising two different 8-color panels: CD3-FITC, CD4-APC / Cy7, CD25-APC, FoxP3-PE, CD8- Cy5-5, Ki67-AmCyan, CD45RA-PE / Cy7, CD39-Pacific Blue, CD45RO-PerCP / Cy5-5, CTLA-4-Pacific Blue, CD127-AmCyan, HLADR-PE / Cy7. Along with Treg immunophenotyping, we identified the paraprotein subtype in newly diagnosed patients and assessed the MRD status with MFC in those MM patients that achieved CR after treatment, thus grouping the latter based on the presence or not of MRD in their bone marrow. Based on the above, we determined the (%) levels of Tregs and studied their functional profile separately in three groups of patients: a) newly diagnosed MM patients, b) MRD+ patients, and c) MRD- patients. Our results revealed that in all three groups of patients (newly diagnosed, MRD+, MRD-) Tregs are numerically higher in the peripheral blood than in the bone marrow. Also, MRD- patients displayed the highest CD3+CD4+CD25hiFoxP3+ Tregs and CD3+CD4+CD25hiFoxP3+CD127lo Tregs, expressed as separate percentages on gated CD4+ T cells and total nucleated cells. The group of newly diagnosed patients was characterized by higher Treg ratio on gated CD4+ T cells in the peripheral blood rather than in the bone marrow, whereas MRD+ and MRD- patients displayed the opposite trend. Concerning the study of the functional profile of CD3+CD4+CD25hiFoxP3+ Tregs there were no differences between bone marrow and peripheral blood in each group; however MRD+ patients showed higher rates of CD39+ Tregs. The above results suggest that Tregs in MRD+ patients, despite not showing an increase in absolute numbers, may have a stronger immunosuppressive phenotype, therefore facilitating the escape of myeloma cells from immunosurveillance. It is also possible that the drugs used in MM therapeutics directly or indirectly alter the migration capacity and anchoring pattern of Tregs from the peripheral blood to the bone marrow or vice versa. Nevertheless, more samples need to be analyzed in order to decipher the exact role of Tregs in the pathophysiology of MM

Deep Phenotyping Reveals Distinct Immune Signatures Correlating with Prognostication, Treatment Responses, and MRD Status in Multiple Myeloma

Despite recent advances, Multiple Myeloma (MM) remains an incurable disease with apparent heterogeneity that may explain patients’ variable clinical outcomes. While the phenotypic, (epi)genetic, and molecular characteristics of myeloma cells have been thoroughly examined, there is limited information regarding the role of the bone marrow (BM) microenvironment in the natural history of the disease. In the present study, we performed deep phenotyping of 32 distinct immune cell subsets in a cohort of 94 MM patients to reveal unique immune profiles in both BM and peripheral blood (PB) that characterize distinct prognostic groups, responses to induction treatment, and minimal residual disease (MRD) status. Our data show that PB cells do not reflect the BM microenvironment and that the two sites should be studied independently. Adverse ISS stage and high-risk cytogenetics were correlated with distinct immune profiles; most importantly, BM signatures comprised decreased tumor-associated macrophages (TAMs) and erythroblasts, whereas the unique Treg signatures in PB could discriminate those patients achieving complete remission after VRd induction therapy. Moreover, MRD negative status was correlated with a more experienced CD4- and CD8-mediated immunity phenotype in both BM and PB, thus highlighting a critical role of by-stander cells linked to MRD biology

Management of Acute Radiodermatitis in Non-Melanoma Skin Cancer Patients Using Electrospun Nanofibrous Patches Loaded with Pinus halepensis Bark Extract

Acute radiodermatitis is the most common side effect in non-melanoma skin cancer patients undergoing radiotherapy. Nonetheless, despite the ongoing progress of clinical trials, no effective regimen has been found yet. In this study, a non-woven patch, comprised of electrospun polymeric micro/nanofibers loaded with an aqueous extract of Pinus halepensis bark (PHBE), was fabricated and clinically tested for its efficacy to prevent radiodermatitis. The bioactivity of the PHBE patch was evaluated in comparison with a medical cream indicated for acute radiodermatitis. Twelve volunteer patients were selected and randomly assigned to two groups, applying either the PHBE patch or the reference cream daily. Evaluation of radiation-induced skin reactions was performed during the radiotherapy period and 1 month afterwards according to the Radiation Therapy Oncology Group (RTOG) grading scale, photo-documentation, patient-reported outcomes (Visual Analog Scale, questionnaire), biophysical measurements (hydration, transepidermal water loss, erythema, melanin), and image analysis. In contrast with the reference product, the PHBE patch showed significant anti-inflammatory activity and restored most skin parameters to normal levels 1 month after completion of radiation therapy. No adverse event was reported, indicating that the application of the PHBE patch can be considered as a safe medical device for prophylactic radiodermatitis treatment

Deep Phenotyping Reveals Distinct Immune Signatures Correlating with Prognostication, Treatment Responses, and MRD Status in Multiple Myeloma

Simple Summary In Multiple Myeloma (MM) malignant cells accumulate in the bone marrow (BM), where they interact with various cell populations. These complex interactions impose mechanisms of tumor growth and proliferation, immune surveillance and immune evasion. The aim of the present study was a detailed immune characterization of MM during the course of the disease, in order to highlight signatures which are clinically relevant. Analyses of both BM and peripheral blood (PB) in matched patients' samples, we showed that PB cannot representatively reflect the BM microenvironment. Particular immune signatures in BM and PB significantly correlated with established prognostic features and could independently associate with distinct responses to the same induction therapy. Moreover, our data provide evidence of a diverse immune profile according to patients' MRD status post treatment. Finally, we provide insights that unique PB immune profiles may be used for the prediction of MRD status through a simple non-invasive approach. Despite recent advances, Multiple Myeloma (MM) remains an incurable disease with apparent heterogeneity that may explain patients' variable clinical outcomes. While the phenotypic, (epi)genetic, and molecular characteristics of myeloma cells have been thoroughly examined, there is limited information regarding the role of the bone marrow (BM) microenvironment in the natural history of the disease. In the present study, we performed deep phenotyping of 32 distinct immune cell subsets in a cohort of 94 MM patients to reveal unique immune profiles in both BM and peripheral blood (PB) that characterize distinct prognostic groups, responses to induction treatment, and minimal residual disease (MRD) status. Our data show that PB cells do not reflect the BM microenvironment and that the two sites should be studied independently. Adverse ISS stage and high-risk cytogenetics were correlated with distinct immune profiles; most importantly, BM signatures comprised decreased tumor-associated macrophages (TAMs) and erythroblasts, whereas the unique Treg signatures in PB could discriminate those patients achieving complete remission after VRd induction therapy. Moreover, MRD negative status was correlated with a more experienced CD4- and CD8-mediated immunity phenotype in both BM and PB, thus highlighting a critical role of by-stander cells linked to MRD biology

Prothymosin α and Its C-Terminal Immunoreactive Decapeptide Show no Evidence of Acute Toxicity: A Preliminary in Silico, in Vitro and in Vivo Investigation.

BACKGROUND: Members of the α-thymosin family have long been studied for their immunostimulating properties. Among them, the danger-associated molecular patterns (DAMPs) prothymosin α (proTα) and its C-terminal decapeptide proTα(100-109) have been shown to act as immunomodulators in vitro, due to their ability to promote T helper type 1 (Th1) responses. Recently, we verified these findings in vivo, showing that both proTα and proTα(100-109) enhance antitumor-reactive T cell-mediated responses. METHODS: In view of the eventual use of proTα and proTα(100-109) in humans, we investigated their safety profile in silico, in human leukocytes and cancer cells lines in vitro, and in immunocompetent mice in vivo, in comparison to the proTα derivative thymosin alpha 1 (Τα1), a 28-mer peptide extensively studied for its safety in clinical trials. RESULTS: In silico prediction via computational tools showed that all three peptide sequences likely are non-toxic or do not induce allergic regions. In vitro, proTα, proTα(100-109) and Tα1 did not affect the viability of human cancer cell lines and healthy donor-derived leukocytes, did not promote apoptosis or alter cell cycle distribution. Furthermore, mice injected with proTα, proTα(100-109) and Tα1 at doses equivalent to the suggested dose regimen of Tα1 in humans, did not show signs of acute toxicity, whereas proTα and proTα(100-109) increased the levels of proinflammatory and Th1-type cytokines in their peripheral blood. CONCLUSION: Our preliminary findings suggest that proTα and proTα(100-109), even at high concentrations, are non-toxic in vitro and in an acute toxicity model in vivo; moreover, we show that the two peptides retain their immunomodulatory properties in vivo and, eventually, could be considered for therapeutic use in humans