Cell Epitope Specificity Research Articles

Comprehensive epitope mapping using polyclonally expanded human CD8 T cells and a two-step ELISpot assay for testing large peptide libraries

The genetic diversity of circulating HIV-1 strains poses a major barrier to the design, development and evaluation of HIV-1 vaccines. The assessment of both vaccine- and natural infection-elicited T cell responses is commonly done with multivalent peptides that are designed to maximally capture the diversity of potential T cell epitopes (PTEs) observed in natural circulating sequences. However, depending on the sequence diversity of viral subtypes and number of the HIV immunogens under investigation, PTE estimates, including HLA-guided computational methods, can easily generate enormous peptide libraries. Evaluation of T cell epitope specificity using such extensive peptide libraries is usually limited by sample availability, even for high-throughput and robust epitope mapping techniques like ELISpot assays. Here we describe a novel, two-step protocol for in-vitro polyclonal expansion of CD8 T cells from a single vial of frozen PBMC, which facilitated the screening 441 HIV-1 Gag peptides for immune responses among 32 HIV-1 positive subjects and 40 HIV-1 negative subjects for peptide qualification. Using a pooled-peptide mapping strategy, epitopes were mapped in two sequential ELISpot assays; the first ELISpot screened 33 large peptide pools using CD8 T cells expanded for 7 days, while the second step tested pool-matrix peptides to identify individual peptides using CD8 T cells expanded for 10 days. This comprehensive epitope screening established the breadth and magnitude of HIV-1 Gag-specific CD8 T cells and further revealed the extent of immune responses to variable/polymorphic epitopes.

Protein Vaccination Directs the CD4+ T Cell Response toward Shared Protective Epitopes That Can Be Recalled after Influenza Virus Infection.

Vaccination is widely used to generate protective immunity against influenza virus. CD4+ T cells contribute in diverse ways to protective immunity, most notably, in the provision of help for the production of neutralizing antibodies. Several recent reports have suggested that influenza virus infection elicits CD4+ T cells whose specificity only partially overlaps that of T cells elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza virus vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4+ T cell repertoire, we evaluated protein vaccine- versus infection-generated CD4+ T cell epitopes. Our studies revealed that all the infection-elicited CD4+ T cell epitope specificities are also elicited by protein vaccination, although the immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4+ memory T cells are recalled and boosted after infection and provide early help to accelerate hemagglutinin (HA)-specific antibody responses. The early CD4+ T cell response and HA-specific antibody production are associated with lowered viral titers during the infection challenge. Our data lend confidence to the ability of current protein-based vaccines to elicit influenza virus-specific CD4+ T cells that can potentiate protective immunity upon influenza virus infection.IMPORTANCE Most current and new influenza vaccine candidates consist of a single influenza virus protein or combinations of influenza virus proteins. For these vaccines to elicit CD4+ T cells that can be recalled after infection, the peptide epitopes should be shared between the two modes of confrontation. Recently, questions regarding the relatedness of epitope selection by influenza virus infection and protein vaccination have been raised. However, the studies reported here show that the specificity of CD4+ T cells elicited by protein-based vaccines overlaps that of T cells elicited by infection and that CD4+ T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection.

Clinical and Immunological Profile of Anti-factor H Antibody Associated Atypical Hemolytic Uremic Syndrome: A Nationwide Database.

Background: Atypical hemolytic uremic syndrome (aHUS), an important cause of acute kidney injury (AKI), is characterized by dysregulation of the alternative complement pathway. Autoantibodies to factor H (FH), a chief regulator of this pathway, account for a distinct subgroup. While high anti-FH titers predict relapse, they do not correlate well with disease activity and their functional characterization is required.Methods: Of 781 patients <18-year-old of aHUS in the nationwide database from 2007 to 2018, 436 (55.8%) had anti-FH antibodies. Clinical features and outcome of patients managed in the last 6-year (n = 317) were compared to before (n = 119). In plasma samples of 44 patients, levels of serial circulating FH immune complexes (CIC), free FH, soluble terminal complement complex (sC5b-9), sheep red blood cell (SRBC) lysis and epitope specificity (n = 8) were examined. Functional renal reserve, ambulatory hypertension, left ventricular hypertrophy (LVH), and proteinuria were evaluated in a subset.Results: Patients presented with markedly elevated anti-FH titers (10,633.2 ± 998.5 AU/ml). Management varied by center, comprising plasma exchange (PEX; 77.5%) and immunosuppression (73.9%). Patients managed in the last 6-year showed better renal survival at mean 28.5 ± 27.3 months (log rank P = 0.022). Mean anti-FH titers stayed 700–1,164 AU/ml during prolonged follow-up, correlating with CIC. Patients with relapse had lower free-FH during remission [Generalized estimating equations (GEE), P = 0.001]; anti-FH levels ≥1,330 AU/ml and free FH ≤440 mg/l predicted relapse (hazards ratio, HR 6.3; P = 0.018). Epitope specificity was similar during onset, remission and relapse. Antibody titer ≥8,000 AU/ml (HR 2.23; P = 0.024), time to PEX ≥14 days (HR 2.09; P = 0.071) and PEX for <14 days (HR 2.60; P = 0.017) predicted adverse renal outcomes. Combined PEX and immunosuppression improved long-term outcomes (HR 0.37; P = 0.026); maintenance therapy reduced risk of relapses (HR 0.11; P < 0.001). At 4.4±2.5 year, median renal reserve was 15.9%; severe ambulatory, masked and pre-hypertension were found in 38, 30, and 18%, respectively. Proteinuria and LVH occurred in 58 and 28% patients, respectively.Conclusion: Prompt recognition and therapy with PEX and immunosuppression, is associated with satisfactory outcomes. Free-FH predicts early relapses in patients with high anti-FH titers. A significant proportion of impaired functional reserve, ambulatory hypertension, proteinuria and LVH highlight the need for vigilant long-term follow-up.

CD4 T Cell Epitope Specificity and Cytokine Potential Are Preserved as Cells Transition from the Lung Vasculature to Lung Tissue following Influenza Virus Infection

Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza virus infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza virus infection in conjunction with intravascular labeling in vivo, the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency, and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue.IMPORTANCE CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood, and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as they establish residence in the lung. However, this transition does not edit CD4 T cell epitope specificity or the cytokine potential of the CD4 T cells. Thus, CD4 T cells that enter the infected lung can convey diverse functions and have a sufficiently broad viral antigen specificity to detect the complex array of infected cells within the infected tissue, offering the potential for more effective protective function.

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.

β2-Glycoprotein I-specific T Cells Are Associated with Epitope Spread to Lupus-related Autoantibodies

Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein β2-glycoprotein I (β2GPI). Notably, mice immunized with β2GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited β2GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that β2GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their β2GPI T cell epitope specificity or MHC class II haplotype. Although β2GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different β2GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one β2GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403(+) autoimmune patient. These data suggest that the generation of a β2GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a β2GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.

Superior Control of HIV-1 Replication by CD8+ T Cells Targeting Conserved Epitopes: Implications for HIV Vaccine Design

A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8+ T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8+ T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8+ T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8+ T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009). Moreover, subjects possessing CD8+ T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021). The association between viral control and the breadth of conserved CD8+ T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215). The associations with viral control were independent of functional avidity of CD8+ T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8+ T cell responses to multiple conserved epitopes of HIV-1.

Breadth or conservation score (CS): which is more important for HIV-1 T cell based vaccine immunogen design?

Background One of the greatest challenges to develop an efficacious HIV vaccine is the enormous diversity of HIV-1. To tackle this problem, T cells based vaccine approaches have come up with two main camps: the mosaic immunogen camp: increasing the breadth of vaccine-induced responses, and the conserved immunogen camp: targeting vaccineinduced T-cell responses only to highly conserved viral regions. While both approaches are theoretically sound, there is no current data suggesting that either approach will be successful in inducing T cells with superior antiviral efficacy. Here we analyzed T cell responses elicited during early HIV-1 infection, to address the question whether CS of targeted epitopes and breadth of T cell responses play an important role in viral control. Methods Using IFN-g ELISpot, we comprehensively mapped T cell epitope specificities recognized by 24 ART-naive individuals during early infection. We identified CS of targeted epitopes, where the CS is defined as the proportion of random HIV-1 group M amino acid sequences in the LANL database that include the epitope. We used a prediction model to impute the viral load (VL) set-point using the first available VL as a predictor for subjects lacking VL set-point. We further evaluated the association between the CS of the targeted epitopes and breadth of T cell responses to the individuals’ VL set-point.

T Cell Epitope Specificity and Pathogenesis of Mouse Hepatitis Virus-1–Induced Disease in Susceptible and Resistant Hosts

Intranasal mouse hepatitis virus-1 (MHV-1) infection of susceptible mouse strains mimics some important pathologic features observed in the lungs of severe acute respiratory syndrome (SARS)-coronavirus-infected humans. The pathogenesis of SARS remains poorly understood, although increasing evidence suggests that immunopathology could play an important role. We previously reported that the adaptive immune response plays an important protective role in MHV-1-infected resistant B6 mice and that both CD4 and CD8 T cells play a significant role in the development of morbidity and lung pathology following intranasal MHV-1 infection of susceptible C3H/HeJ and A/J mice. In this study, we have identified novel CD4 and CD8 epitopes in MHV-1-infected susceptible and resistant strains of mice. Susceptible C3H/HeJ mice mount robust and broad MHV-1-specific CD4 T cell responses, whereas in resistant B6 mice, Ag-specific CD8 T cell responses dominate. We also show that previously immunized susceptible C3H/HeJ mice do not develop any morbidity and are completely protected following a lethal-dose MHV-1 challenge despite mounting only a modest secondary T cell response. Finally, we demonstrate that the resistance displayed by B6 mice is not solely accounted for by the elaboration of a broad and vigorous MHV-1-specific CD8 T cell response, as MHV-1 infection of C3.SW-H2(b)/SnJ mice, which mount an equally robust CD8 T cell response of the same specificity, is still associated with significant morbidity. Thus, identification of novel CD4 and CD8 T cell epitopes for MHV-1 permitted high-resolution analyses of pulmonary T cell responses in a mouse model of SARS.

Epitope Mapping of HIV-Specific CD8+ T cells in a Cohort Dominated by Clade A1 Infection

BackgroundCD8+ T cell responses are often detected at large magnitudes in HIV-infected subjects, and eliciting these responses is the central aim of many HIV-1 vaccine strategies. Population differences in CD8+ T cell epitope specificity will need to be understood if vaccines are to be effective in multiple geographic regions.Methodology/Principal FindingsIn a large Kenyan cohort, we compared responsive CD8+ T cell HIV-1 Env overlapping peptides (OLPs) to Best Defined Epitopes (BDEs), many of which have been defined in clade B infection. While the majority of BDEs (69%) were recognized in this population, nearly half of responsive OLPs (47%) did not contain described epitopes. Recognition frequencies of BDEs were inversely correlated to epitopic sequence differences between clade A1 and BDE (P = 0.019), and positively selected residues were more frequent in “new” OLPs (without BDEs). We assessed the impact of HLA and TAP binding on epitope recognition frequencies, focusing on predicted and actual epitopes in the HLA B7 supertype.Conclusions/SignificanceAlthough many previously described CD8 epitopes were recognized, several novel CD8 epitopes were defined in this population, implying that epitope mapping efforts have not been completely exhausted. Expansion of these studies will be critical to understand population differences in CD8 epitope recognition.

HLA-DR Alleles in Amyloid β-Peptide Autoimmunity: A Highly Immunogenic Role for the DRB1*1501 Allele

Active amyloid beta-peptide (Abeta) immunization of patients with Alzheimer's disease (AD) caused meningoencephalitis in approximately 6% of immunized patients in a clinical trial. In addition, long-term studies of AD patients show varying degrees of Abeta Ab responses, which correlate with the extent of Abeta clearance from the brain. In this study, we examined the contribution of various HLA-DR alleles to these immune-response variations by assessing Abeta T cell reactivity, epitope specificity, and immunogenicity. Analysis of blood samples from 133 individuals disclosed that the abundant DR haplotypes DR15 (found in 36% of subjects), DR3 (in 18%), DR4 (12.5%), DR1 (11%), and DR13 (8%) were associated with Abeta-specific T cell responses elicited via distinct T cell epitopes within residues 15-42 of Abeta. Because the HLA-DRB1*1501 occurred most frequently, we examined the effect of Abeta challenge in humanized mice bearing this allele. The observed T cell response was remarkably strong, dominated by secretion of IFN-gamma and IL-17, and specific to the same T cell epitope as that observed in the HLA-DR15-bearing humans. Furthermore, following long-term therapeutic immunization of an AD mouse model bearing the DRB1*1501 allele, Abeta was effectively cleared from the brain parenchyma and brain microglial activation was reduced. The present study thus characterizes HLA-DR alleles directly associated with specific Abeta T cell epitopes and demonstrates the highly immunogenic properties of the abundant allele DRB1*1501 in a mouse model of AD. This new knowledge enables us to explore the basis for understanding the variations in naturally occurring Abeta-reactive T cells and Abeta immunogenicity among humans.

The use of factor VIII/von Willebrand factor concentrate for immune tolerance induction in haemophilia A patients with high‐titre inhibitors: association of clinical outcome with inhibitor epitope profile

A retrospective chart review of 11 subjects with severe haemophilia A and high-titre inhibitors who received a von Willebrand factor-containing FVIII concentrate (VWF/FVIII) for immune tolerance induction (ITI) was accompanied by B cell inhibitor epitope mapping during 10/11 treatment courses. ITI was successful or partially successful in all seven subjects who received VWF/FVIII for initial ITI, and failed in all four subjects whose ITI with this product was initiated following treatment failure using recombinant factor VIII. Variables including age at inhibitor development and age at ITI initiation, interval between inhibitor detection and ITI initiation, titre at start of ITI, and peak historical titres prior to and during ITI were not statistically significant outcome predictors in this cohort. However, the B cell epitope specificity in all four successful and in one of two partially successful ITI subjects for whom information was available included the C2 and excluded the A2 domains. Conversely, FVIII B cell epitopes in one partially successful ITI and in all three failed ITI subjects for whom data were available mapped to both the C2 and the A2 domains. The FVIII B cell epitope profile was associated with ITI outcome in this VWF/FVIII-treated cohort. Its role in predicting ITI outcome and guiding choice of FVIII product for ITI requires further study.

Role of Human Leucocyte Antigen DQ in the Presentation of T Cell Epitopes in the Major Cow’s Milk Allergen αs1-Casein

Background: Little is known about the association between human leucocyte antigen (HLA) and cow’s milk allergy (CMA). The aim of the present study was to determine the HLA restriction of T cell clones (TCCs) specific to αs1-casein, the most abundant milk protein, and to study possible HLA class II allele associations with CMA. Methods: αs1-Casein-specific TCCs were derived from 6 children with CMA, 9 atopic children without CMA and 5 non-atopic children. T cell epitope specificity was defined by stimulation with overlapping peptides, spanning the αs1-casein molecule. HLA restriction was determined in proliferation assays using antibodies blocking either HLA-DP, HLA-DQ or HLA-DR. HLA genotyping was performed in 32 subjects with CMA, 23 atopic and 22 non-atopic individuals. Results: Ten TCCs were restricted to HLA-DQ, 6 TCCs to HLA-DR and 4 TCCs to HLA-DP. The sequence in αs1-casein that was most immunogenic to T cells from children with CMA contained T cell epitopes restricted to DQB1*0201, DPB1*0401 and DRB1*1501. The DQB1*0501 allele frequency was lower in children with CMA than in non-atopic children, but this difference could not be confirmed in an additional group of subjects with and without CMA. Conclusions: HLA-DQ plays a substantial role in the presentation of T cell epitopes in αs1-casein. However, HLA class II allele frequencies do not show major differences between cow’s milk allergic, atopic and non-atopic subjects. T cell epitopes in the most immunogenic region are presented by various abundantly present HLA genotypes. Therefore, this sequence may be a suitable target for peptide immunotherapy.

T Cell Epitope Mimicry in Antiglomerular Basement Membrane Disease

Antiglomerular basement membrane (GBM) disease or Goodpasture's syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4alpha3 NC1 (Col4alpha3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28-40 of Col4alpha3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28-40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28-40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16-25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.

T cell mimicry and epitope specificity of cross-reactive T cell clones from rheumatic heart disease.

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein-specific human T cell clones derived from rheumatic carditis were cross-reactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4(+) and CD8(+) cross-reactive T cell clones, at least 6 different reactivity patterns were distinguished, suggesting different degrees of cross-reactivity and a very diverse T cell repertoire. The latter was confirmed by a heterogeneous Vbeta gene and CDR3 usage. HLA restriction and Th1 cytokine production in response to rM6 protein were preserved when the T cell clones were stimulated by human cardiac myosin or other alpha-helical proteins, such as tropomyosin and laminin. The cross-reactive human T cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin regions. In our study, T cell mimicry was defined as recognition of structurally related Ags involved in disease and recognized by the same T cell. Mimicry in our study was related to alpha-helical coiled coil proteins which have a repetitive seven-aa residue periodicity that maintains alpha-helical structure and thus creates a high number of degenerate possibilities for recognition by T cells. The study of human T cell clones from rheumatic heart disease revealed potential sites of T cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T cell mimicry in human autoimmune disease.

B cell epitope specificity in ANCA-associated vasculitis: does it matter?

Pauci-immune idiopathic small-vessel vasculitis is strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA). Antibodies to PR3 predominate in patients with Wegener's granulomatosis; antibodies to myeloperoxidase (MPO) are found more frequently in patients with microscopic polyangiitis. There is increasing in vivo and in vitro evidence for a pathogenic role of ANCA in systemic vasculitis based on associations of ANCA with disease activity. If ANCA are pathogenic, why is the course of disease different from one patient to another? Antibodies can recognize different binding sites (epitopes) on their corresponding antigens. Differences in binding specificity may influence the pathogenic potential of the antibodies. Differences between epitope specificity of ANCA between patients or changes in epitope specificity of ANCA in time in an individual patient may, accordingly, result in differences in disease expression. This review will focus on epitope specificity of autoantibodies in systemic autoimmune diseases and especially on the epitope specificity of PR3- and MPO-ANCA. We will discuss whether PR3-ANCA or MPO-ANCA recognize different epitopes on PR3 and MPO, respectively, and whether the epitopes recognized by ANCA change in parallel with the disease activity of ANCA-associated vasculitis. Finally, we will speculate if the direct pathogenic role of ANCA can be ascribed to one relapse- or disease-inducing epitope. Characterization of relapse- or disease-inducing epitopes bound by PR3-ANCA and MPO-ANCA is significant for understanding initiation and reactivation of ANCA-associated vasculitis. Elucidating a disease-inducing epitope bound by ANCA may lead to the development of epitope-specific therapeutic strategies.

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501.

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.

Marmoset Fine B Cell and T Cell Epitope Specificities Mapped onto a Homology Model of the Extracellular Domain of Human Myelin Oligodendrocyte Glycoprotein

Aberrant association of autoantibodies with myelin oligodendrocyte glycoprotein (MOG), an integral membrane protein of the central nervous system (CNS) myelin, has been implicated in the pathogenesis of multiple sclerosis (MS). Sensitization of nonhuman primates (Callithrix jacchus marmosets) against the nonglycosylated, recombinant N-terminal domain of rat MOG (residues 1–125) reproduces an MS-like disease in which MOG-specific autoantibodies directly mediate demyelination. To assess the interrelationship between MOG structure and the induction of autoimmune CNS diseases and to enable structure-based rational design of therapeutics, a homology model of human MOG(2–120) was constructed based on consensus residues found in immunoglobulin superfamily variable-type proteins having known structures. Possible sites for posttranslational modifications and dimerization have also been identified and analyzed. The B cell and T cell epitopes have been identified in rat MOG-immunized marmosets, and these sequences are observed to map primarily onto accessible regions in the model, which may explain their ability to generate potent antibody responses.

Analysis of T cell epitope specificity differentiating pathogenic and non-pathogenic Theiler's murine encephalomyelitits virus

Analysis of T cell epitope specificity differentiating pathogenic and non-pathogenic Theiler's murine encephalomyelitits virus

T cell epitope specificity in human allergic and nonallergic subjects to bee venom phospholipase A2.

Phospholipase A2 (PLA) is a biochemically fully defined glycoprotein, representing the main allergen of bee venom. We have established CD4+ T cell clones specific to PLA from subjects allergic and nonallergic to bee sting. By screening the epitope specificity of these clones with 62 synthetic overlapping dodecapeptides representing the PLA molecule, two immunogenic epitopes, PLA81-92 and PLA113-124, were identified. Additional screening, using longer peptides of up to 18 residues, revealed a third epitope at position PLA 45-62. These epitopes are recognized by specific T cell clones in association with HLA-DP and -DQ molecules, although HLA-DR-associated responses to PLA exist. Primary in vitro proliferation of unfractionated PBMC from bee sting allergic, hyposensitized, or hyperimmune subjects to PLA-derived peptides revealed the same immunogenic sites as found in the T cell clones. Among the different groups of individuals, proliferative responses to the PLA molecule and fragments thereof were generally higher in allergic patients than in nonallergic subjects. Thus, at least three linear epitopes are involved in T cell recognition of PLA, independently whether or not subjects are allergic.