Prediction of desmoglein-3 peptides reveals multiple shared T-cell epitopes in HLA DR4- and DR6- associated Pemphigus vulgaris

BACKGROUND: Pemphigus vulgaris (PV) is a severe autoimmune blistering skin disorder that is strongly associated with major histocompatibility complex class II alleles DRB1*0402 and DQB1*0503. The target antigen of PV, desmoglein 3 (Dsg3), is crucial for initiating T-cell response in early disease. Although a number of T-cell specificities within Dsg3 have been reported, the number is limited and the role of T-cells in the pathogenesis of PV remains poorly understood. We report here a structure-based model for the prediction of peptide binding to DRB1*0402 and DQB1*0503. The scoring functions were rigorously trained, tested and validated using experimentally verified peptide sequences. RESULTS: High predictivity is obtained for both DRB1*0402 (r(2 )= 0.90, s = 1.20 kJ/mol, q(2 )= 0.82, s(press )= 1.61 kJ/mol) and DQB1*0503 (r(2 )= 0.95, s = 1.20 kJ/mol, q(2 )= 0.75, s(press )= 2.15 kJ/mol) models, compared to experimental data. We investigated the binding patterns of Dsg3 peptides and illustrate the existence of multiple immunodominant epitopes that may be responsible for both disease initiation and propagation in PV. Further analysis reveals that DRB1*0402 and DQB1*0503 may share similar specificities by binding peptides at different binding registers, thus providing a molecular mechanism for the dual HLA association observed in PV. CONCLUSION: Collectively, the results of this study provide interesting new insights into the pathology of PV. This is the first report illustrating high-level of cross-reactivity between both PV-implicated alleles, DRB1*0402 and DQB1*0503, as well as the existence of a potentially large number of T-cell epitopes throughout the entire Dsg3 extracellular domain (ECD) and transmembrane region. Our results reveal that DR4 and DR6 PV may initiate in the ECD and transmembrane region respectively, with implications for immunotherapeutic strategies for the treatment of this autoimmune disease

pDOCK: a new technique for rapid and accurate docking of peptide ligands to Major Histocompatibility Complexes

Background: Identification of antigenic peptide epitopes is an essential prerequisite in T cell-based molecular vaccine design. Computational (sequence-based and structure-based) methods are inexpensive and efficient compared to experimental approaches in screening numerous peptides against their cognate MHC alleles. In structure-based protocols, suited to alleles with limited epitope data, the first step is to identify high-binding peptides using docking techniques, which need improvement in speed and efficiency to be useful in large-scale screening studies. We present pDOCK: a new computational technique for rapid and accurate docking of flexible peptides to MHC receptors and primarily apply it on a non-redundant dataset of 186 pMHC (MHC-I and MHC-II) complexes with X-ray crystal structures. Results: We have compared our docked structures with experimental crystallographic structures for the immunologically relevant nonameric core of the bound peptide for MHC-I and MHC-II complexes. Primary testing for re-docking of peptides into their respective MHC grooves generated 159 out of 186 peptides with Ca RMSD of less than 1.00 Å, with a mean of 0.56 Å. Amongst the 25 peptides used for single and variant template docking, the Ca RMSD values were below 1.00 Å for 23 peptides. Compared to our earlier docking methodology, pDOCK shows upto 2.5 fold improvement in the accuracy and is ~60% faster. Results of validation against previously published studies represent a seven-fold increase in pDOCK accuracy. Conclusions: The limitations of our previous methodology have been addressed in the new docking protocol making it a rapid and accurate method to evaluate pMHC binding. pDOCK is a generic method and although benchmarks against experimental structures, it can be applied to alleles with no structural data using sequence information. Our outcomes establish the efficacy of our procedure to predict highly accurate peptide structures permitting conformational sampling of the peptide in MHC binding groove. Our results also support the applicability of pDOCK for in silico identification of promiscuous peptide epitopes that are relevant to higher proportions of human population with greater propensity to activate T cells making them key targets for the design of vaccines and immunotherapies.16 page(s

Analysis of polymorphism in CD86, CTLA4, TNF and IL10 genes in Serbian patients with pemphigus

Uvod: Oboljenja iz grupe pemfigusa - pemfigus vulgaris (PV) i pemfigus foliaceus (PF) su autiomunska bulozna oboljenja kože i sluznica. U patogenezi su od značaja autoantitela na dezmogleine i druge molekule koja dovode do akantolize i formiranja intraepidermalnog rascepa. Za aktivaciju autoreaktivnih T-ćelija u pemfigusu neophodno je prepoznavanja dezmogleinskih peptida i angažovanje CD28 molekula, koji je najznačajniji kostimulatorni receptor u aktivaciji naivnih T ćelija. CD28 molekul se vezuje za B7 familiju molekula kao što su CD80 i CD86. Pored toga, B7 molekuli mogu da aktiviraju inhibitorni receptor na T-ćelijama, CTLA-4, koji kontroliše mehanizam održavanja tolerancije na sopstvene peptide. Na značaj genetskih faktora u patogenezi pemfigusa ukazuju prethodna istraživanja kojima su definisani određeni genetski faktori kao što su i polimorfizmi pojedinačnih nukleotida u genima molekula od značaja za patogenezu pemfigusa. Ciljevi istraživanja: Odrediti distribuciju alela u genima za molekule CD86, CTLA-4, TNF i IL-10 kod pacijenata sa PV i PF, kao i kod zdravih osoba (dobrovoljni davaoci krvi) u Srbiji, ispitati da li je neki od ispitivanih polimorfizama faktor rizika za nastanak oboljenja (PV i PF) i uporediti ih sa podacima iz literature koji su dobijeni ispitivanjem istih polimorfizama kod geografski i etnički različitih populacija. Pacijenti i metode: Istraživanje je obuhvatilo 61 bolesnika sa pemfigusom od kojih je PV imalo 48, a PF 13 bolesnika i 486 zdravih osoba-kontrola (dobrovoljni davaoci krvi). Dijagnoza pacijenata sa pemfigusom postavljena je na osnovu kliničke slike i testa direktne imunofluorescencije (DIF test). Za detekciju i analizu polimorfizama gena za CD86, CTLA-4, TNF i IL-10 korišćene su komercijalne optimizovane smeše specifičnih oligonukleotida i TaqMan proba obeleženih FAM i VIC fluorohromima i to: CD86 rs1129055, CTLA4 rs733618 i rs5742909, TNF rs1800629 i rs361525 i IL-10 rs1800896 i rs1800871 (PE, Applied Biosystems). Amplifikacija fragmenata DNK Real-time PCR metodom vršena je pomoću navedenih smeša komercijalnih oligonukleotida i Maxima™ Probe qPCR 2X Master Mix, (Fermentas) uzimajući u obzir preporuke proizvođača reagenasa...Introduction: Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are rare autoimmune blistering diseases with presumed T cell dependent pathology. Activation of naïve T cells is dependent on antigen recognition, subsequent signaling through the T cell receptor complex (signal 1), and various other interactions of T cells with antigen presenting cells that may be collectively designated as signal 2, which is unconditionally required for T cell activation both in response to infection and to autoantigens. Among the best described interactions contributing to signal 2 are those mediated by B7 family molecules such as CD80 and CD86 with their ligands, CD28, providing activation signals, and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), conferring inhibition. Single nucleotide polymorphisms (SNPs) within genes encoding those molecules may alter the signaling process. It is not known whether functional genetic polymorphisms within genes encoding the aforementioned proteins may increase risk for developing PV and PF and, if so, whether they might serve as biomarkers for susceptibility to these diseases. Aims of the investigation: To examine functional single nucleotide polymorphisms within CD86 (rs1129055), CTLA4 (rs733618 and rs5742909), TNF (rs1800629 andrs361525), and IL-10 (rs1800896 and rs1800871) genes in 61 pemphigus patients and 486 healthy controls, and to compare results in Serbian pemphigus patients and healthy controls with results obtained in different populations groups. Patients and methods: Our study included 61 pemphigus patients, among them 48 with PV and 13 with PF, treated at the Dermatovenereology Clinics of the Clinical Centre of Serbia. Patients were stratified according to clinical presentation of the disease as a PV or PF patients. Diagnosis in all patients was established by merging clinical findings, histopathologic analysis, and the results of direct immunofluorescence tests. Blood samples from 486 healthy blood donors were obtained from the National Blood Transfusion Institute..

Charakterisierung der adaptiven Autoimmunantwort beim Pemphigus vulgaris

Pemphigus vulgaris (PV) ist eine antikörpervermittelte Autoimmunerkrankung, bei der es zur Bildung von Blasen und Erosionen an der Haut und den Schleimhäuten kommt. IgG-Autoantikörper gegen die desmosomalen Cadherine Desmoglein (Dsg)1 und 3 sind maßgeblich für die Entstehung des PV verantwortlich, indem sie einen Adhäsionsverlust der epidermalen Keratinozyten (Akantholyse) induzieren. Autoreaktiven CD4+ T-Zellen sind entscheidend für die Aktivierung und Differenzierung von autoreaktiven B-Zellen und für die Induktion der Autoantikörperproduktion. Die genauen zellulären und humoralen Mechanismen, die zur Produktion von Autoantikörpern führen, sind in weiten Teilen noch nicht verstanden Ziel dieser Arbeit war es daher, die adaptive Autoimmunantwort beim PV mit Hinblick auf die für die Induktion von Autoantikörpern relevanten Zytokine und die beteiligten T- und B-Zell-Subpopulationen näher zu untersuchen. Hierfür wurden in einer Querschnittsstudie Plasma und Immunzellen aus dem peripheren Blut von insgesamt 24 PV-Patienten untersucht. Die Ergebnisse zeigten eine Rolle von Interleukin (IL)-21, ein pleiotropes Zytokin, welches die Differenzierung von B-Zellen in antikörpersezernierende Plasmazellen induzieren kann, in der Pathogenese des PV auf. Es konnten erhöhte Plasmaspiegel dieses Zytokins im Vergleich zu gesunden Kontrollen detektiert werden, wobei IL-21 vorrangig von CD4+ T-Zellen bei PV Patienten mit aktiver Erkrankung produziert wurde. IL-21-produzierende T-Zellen zeichneten sich in Teilen durch eine Koproduktion der Zytokine IL-4 und IL-17 aus, welches eine partielle Beteiligung von Th2- und Th17-Zellen an der IL-21-Produktion nahelegte. Nach in vitro Stimulation der peripheral blood mononuclear cells (PBMC) von Patienten mit Dsg3 konnte die Produktion von IL-21 erstmals bei autoreaktiven T-Zellen mittels des sensitiven ELISpot-Verfahrens nachgewiesen werden, wodurch die funktionelle Bedeutung dieses Zytokins in der Pathogenese zusätzlich unterstrichen wurde. Des Weiteren lagen zirkulierende T follikuläre Helfer (Tfh)-Zellen (definiert als CD4+CXCR5+ T-Zellen), die eine essentielle Funktion bei der Induktion einer antikörpervermittelten Immunantwort besitzen, im erhöhten Maße im peripheren Blut der Patienten vor. Hinsichtlich der funktionell relevanten kostimulatorischen Moleküle programmed cell death protein 1 (PD-1) und inducible T cell costimulator (ICOS) zeigten Tfh-Zellen von Patienten keine Unterschiede im Vergleich zu gesunden Kontrollen. Darüber hinaus wurden niedrigfrequente autoreaktive B-Zellen mittels eines zuvor etablierten durchflusszytometrischen Testsystems durch die Markierung von rekombinanten Dsg3 mit Alexa Fluor 647 (Dsg3-AF647) im peripheren Blut der Patienten identifiziert und die Dsg3-spezifischen B-Zell-Subpopulationen unreif/naiv (CD19+CD27-), Gedächtnis B-Zellen (CD19+CD27+) und Plasmablasten (CD19+CD27++CD38++) eingehend charakterisiert. Vor allem autoreaktive Gedächtnis B-Zellen konnten bei PV-Patienten in einem erhöhten Maße nachgewiesen werden, welches auf eine gestörte periphere Immuntoleranz an diesem Punkt der B-Zell-Entwicklung unter möglicher Beteiligung von autoreaktiven Tfh-Zellen hinwies. In einer Subgruppenanalyse von 5 PV-Patienten, die zuvor mit dem B-Zell-depletierenden anti-CD20-Antikörper Rituximab behandelt wurden, zeigten sich erhöhte Frequenzen von Dsg3-spezifische Gedächtnis B-Zellen insbesondere in Phasen einer klinischen Remission, während dies bei einem Rezidiv nicht beobachtet werden konnten. Es wurde daher vermutet, dass die Aktivierung von autoreaktiven Gedächtnis B-Zellen einen Ausgangspunkt für die erneute Produktion von Autoantikörpern darstellen kann. Die Ergebnisse der vorliegenden Arbeit identifizieren IL-21 und IL-21-produzierende T-Zellen als wichtige Bestandteile in der Pathogenese des PV. Zusammen mit der eingehenden Charakterisierung von Dsg3-spezifischen B-Zell-Subpopulation können diese Erkenntnisse die zukünftige Entwicklung neuer zielgerichteter Therapieformen zur Behandlung des PV ermöglichen

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease