HLA-DR Specificities Research Articles

Precise identification and tracking of HMGCR-reactive CD4+ T cells in the target tissue of patients with anti-HMGCR immune-mediated necrotising myopathy

BackgroundAnti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR)-positive immune-mediated necrotising myopathy (IMNM) is characterised by the presence of IgG autoantibodies against HMGCR and a strong association with specific HLA-DR alleles. Although these findings...

Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis.

BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs.CONCLUSIONAutoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.FUNDINGSupported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.

Analysis of Human Leukocyte Antigen DR Alleles, Immune-Related Adverse Events, and Survival Associated With Immune Checkpoint Inhibitor Use Among Patients With Advanced Malignant Melanoma

Treatment with immune checkpoint inhibitors (ICIs) has increased survival in patients with advanced malignant melanoma but can be associated with a wide range of immune-related adverse events (irAEs). The role of human leukocyte antigen (HLA)-DR alleles in conferring irAE risk has not been well studied. To evaluate the association between irAEs and treatment response, survival, and the presence of HLA-DR alleles after ICI therapy in advanced melanoma. This case-control study used the patient registry and biobanked samples from the tertiary referral University of Colorado Cancer Center. Specimens and clinical data were collected between January 1, 2010, and December 31, 2021. Patients with advanced (stage III unresectable and stage IV) melanoma who received ICI therapy (n = 132) were included in the analysis. Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4, anti-programmed cell death protein 1 or its ligand, or the combination) for the treatment of advanced melanoma. The association between irAEs and response to therapy, survival, and HLA-DR alleles. Among the cohort of 132 patients with advanced melanoma (mean [SD] age, 63.4 [7.2] years; 85 men [64%] and 47 women [36%]) treated with ICIs, 73 patients had at least 1 irAE and 59 did not have an irAE. Compared with patients without an irAE, patients with an irAE had higher treatment response rates (50 of 72 [69%] vs 28 of 57 [49%]; P = .02) and increased survival (median, 4.8 [IQR, 0.2-9.6] vs 3.2 [IQR, 0.1-9.2] years; P = .02). Specific HLA-DR alleles were associated with the type of irAE that developed: 7 of 10 patients (70%) who developed type 1 diabetes had DR4; 6 of 12 (50%) who developed hypothyroidism had DR8; 5 of 8 (63%) who developed hypophysitis had DR15; 3 of 5 (60%) who developed pneumonitis had DR1; and 8 of 15 (53%) who developed hepatitis had DR4. These findings suggest that IrAEs are associated with treatment response rates and increased survival after ICI therapy for advanced melanoma. Because distinct HLA-DR alleles are associated with given adverse events, HLA genotyping before ICI therapy may aid in identifying risk for specific irAEs that could develop with such treatment.

Immunophenotypic Characterisation Along with Aberrant Expression of CD markers in Morphologically Diagnosed Cases of Acute Myeloid Leukemia

Immunophenotyping of leukemia cells is useful for detecting leukemia cell line, determining maturation stage and identifying aberrant antigens which act for individual treatment monitoring and detection of residual disease. A total of 104 newly diagnosed cases of acute myeloid leukemia were identified at hematology department in National Institute of Cancer Research and Hospital from January 2020 to December 2021.We detect Immunophenotypic pattern in newly diagnosed cases of acute myeloid leukemia. We also determine the frequency and pattern of aberrant expression of CD markers in acute myeloid leukemia patients. Mean age of patients was 35 years (SD±16 years) with male to female ratio was 1.53:1. Most frequent morphologic subtype was AMLM2 constituting 33.6% of all AML cases. Lineage specific markers HLADR, CD13, CD33, MPO, CD117 and CD34 were expressed in 80%, 89%, 95%, 77%, 74% and 62% cases of all AML cases respectively. Among 104 AML patient, aberrant CD expression was observed in 36% cases. The most frequently observed aberrant markers were CD7 and CD19 lymphoid markers, that were expressed in 15.38% and 14.42% cases respectively. Less frequent aberrant cCD3, CD10, CD5 and cCD79a antigens were expressed in 2.88%, 1.92%, 0.96% and 0.96% cases respectively. Immunophenotyping is essential in diagnosis and sub-classification of AML and expression of aberrant CD antigens is common in acute myeloid leukemia. These findings suggest the necessity for a more extensive study to evaluate the prognostic significance of aberrant CD marker expression in AML and to improve the accuracy of diagnosis and classification of AML. J MEDICINE 2022; 23: 106-111

Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant.

Regulatory T cells are an important component of an immune response shaping the overall behavior to potential antigens including alloantigens. Multiple mechanisms have been shown to contribute towards developing and sustaining a immunological regulatory response. One of the described contact dependent suppressive mechanisms regulatory cells have been shown to utilize is through the production of adenosine from extracellular ATP mediated by CD39 and CD73. In this study we demonstrate that the adenosinergic pathway plays a major role in the suppressive/regulatory effects antigen specific regulatory T cell enriched lines (ASTRLs) that have been of expanded ex vivo from stable kidney transplant patients. We have previously shown that these ASTRL cells are capable of suppressing alloimmune responses in vitro and significantly prolonging allograft survival in an animal model of kidney transplantation. For this study nineteen ASTRLs were expanded from 17 kidney transplant patients by repeated stimulation of recipient peripheral blood mononuclear cells with donor specific HLA-DR peptides. All 19 ASTRLs showed upregulation of numerous markers associated with regulatory cells and were able to inhibit donor antigen specific T cell proliferation in a dose dependent fashion. ASTRLs suppressed indirect and direct alloimmune responses compatible with our previous animal study findings. Upregulation of both CD39 and CD73 was observed post expansion and ASTRLs demonstrated extracellular hydrolysis of ATP, indicating functionality of the upregulated proteins. We also showed that inhibition of the adenosinergic pathway using inhibitors of CD39 resulted in abrogation of suppression and increased antigen specific T cell proliferation. This demonstrates that the main mechanism of action of the suppressive activity donor peptide driven ASTRLs generated from kidney transplant patients is the adenosinergic pathway. Furthermore this suggests the possibility that combining infusion of Tregs with other treatments, such as adenosine receptor agonists or increasing CD39 expression in the grafts may further enhance a regulatory response to the allograft and possibly achieve transplantation tolerance.

P14.20 Multi-Spectral Imaging of Lung Adenocarcinoma Reveals Importance of Cancer Specific HLA-DR on the TME and Clinical Outcome

Within the tumor microenvironment (TME), the interactions between cancer cells and immune cells are of increasing importance as targeting key immune checkpoints have redefined clinical care for patients with lung cancer. While immune checkpoint inhibitors have improved outcomes for patients with NSCLC, only a subset of patients respond. Tremendous efforts have been made to define factors that mediate tumor progression and response to immunotherapy. Regulation of antigen-presentation has been shown to play a critical role in this response. MHCII (HLA-DR) is normally expressed on antigen presenting cells and leads to activation of CD4+ T cells. Notably, HLA-DR expression is observed on cancer cells and appears to shape local anti-tumor immunity through potential direct presentation of tumor neoantigens to CD4+ helper T cells. While several studies have demonstrated that HLA-DR on cancer cells leads to more T cell infiltration, its effect on the TME and outcomes for patients with NSCLC have not been systemically examined. Advances in multispectral immunohistochemistry techniques allow for quantitative and spatial identification of multiple immune cells. Imaging with multiple markers can reveal the diversity of the local immune infiltrate, cell-cell interactions, and correlations of spatial distribution and location of immune cells in NSCLC with clinical outcome of patients with cancer cells. Slides from 153 patients with resected lung adenocarcinoma were stained using Opal multiplex with a panel of markers: DAPI, HLA-DR, CK, CD3, CD14, CD19 and CD8. Multispectral imaging was performed using wide field microscopy on the Vectra3.0. For each patient, 3-5 regions of interest (ROIs) were chosen and spectral unmixing was utilized for the detection and separation of seven different fluorescent signals in each NSCLC tissue samples. Cells were segmented based on nuclear and membrane markers and classified in phenotypes using inForm machine learning algorithms. The immunophenotypes were quantified and nearest neighbor analysis was performed. The presence of HLA-DR on cancer cells and immune cell infiltration were correlated with clinical outcome. Co-expression of HLA-DR and CK+ defined cancer cells that express MHCII. HLA-DR high and HLA-DR low patients were stratified using a cutoff of >5% of cancer cells expressing HLA-DR. Patients with HLA-DR high tumors had significantly increased immune cell infiltration of CD4+, CD8+, and CD14+ cells. Nearest neighbor analysis demonstrated CK+ tumor cells in HLA-DR high patients were in closer proximity to CD14+, CD19+, CD4+, and CD8+ T cells. In HLA-DR high patients, more CK+ cancer cells were directly interfaced with CD4+ and CD8+ T cells. Patients with high levels of cancer cell HLA-DR experienced improved overall survival at 5 years (p=0.046). HLA-DR high tumors are significantly associated with increased T cell and myeloid cell infiltration, resulting in closer proximity of all immune cells and more cell-cell contact. Imaging analysis of HLA-DR high tumors demonstrated more direct contact with CD4+ and CD8+ T cells compared to HLA-DR low tumors. Patients with HLA-DR high tumors experienced an improved overall survival compared to patients with HLA-DR low tumors. These data suggest cancer cell HLA-DR may represent a new biomarker for TME inflammation and patient outcomes in NSCLC.

Comparison of stem cell markers on Gingival mesenchymal stem cells among diabetic and healthy individuals. An Ex Vivo Pilot study

Aim: Diabetes mellitus (DM), being a risk factor for periodontal disease, affects various cellular functions, and it plays an important role in periodontal regeneration. However, the effect of hyperglycemia on Gingival mesenchymal stem cells (GMSCs) is unclear. We aim at investigating the effect of hyperglycemia on stem cell markers of GMSCs compared with normoglycemic conditions. Materials and Methods: An ex vivo pilot study was conducted by obtaining primary gingival tissues from subjects. Subjects from the outpatient department of periodontology were randomly selected according to the criteria. Three subjects with an HbA1c of >6.5 were selected as the test group, and three subjects with an HbA1c <6 served as controls. The tissue was enzymatically digested in 0.5 mg/mL collagenase (Sigma-Aldrich) and cultured in Knockout Dulbecco‘s Modified Eagles Medium (DMEM - KO medium, Life Technologies). MSC-like cells isolated were examined under a microscope and investigated for specific cell surface antigens CD73, CD90, CD105, CD34, CD45, HLA ABC, and HLA DR by using flow cytometry. Data were analyzed by a two-tailed non-parametric Mann–Whitney U test using SPS software. P < 0.05 was considered statistically significant. Results: The immunophenotype characterization in both test and control exhibited positive expression of CD73 (94.95%,95.93%), CD90 (98.05%, 88.53%), CD105 (73.97%, 73.88%), and HLA ABC (98.83%,98.16%) and negative expression of CD34 (0.02%,0.10%), CD45(0.57%,1.72%), and HLA DR (0.64%, 2.57%). Statistical analysis by the nonparametric Mann–Whitney U test did not reveal any statistically significant difference in the expression. Conclusion: GMSCs from the hyperglycemic environment retained their stem cell characteristics by positive expression of CD73, CD90, CD105, and HLA ABC, and negative expression of CD34, CD45, and HLA DR.

Human Leukocyte Antigen-DR Deficiency and Immunosuppression-Related End-Organ Failure in SARS-CoV2 Infection.

Human Leukocyte Antigen-DR Deficiency and Immunosuppression-Related End-Organ Failure in SARS-CoV2 Infection.

Persistence of dysfunctional natural killer cells in adults with high-functioning autism spectrum disorders: stigma/consequence of unresolved early infectious events?

BackgroundAutism spectrum disorders (ASD) are characterized by abnormal neurodevelopment, genetic, and environmental risk factors, as well as immune dysfunctions. Several lines of evidence suggest alterations in innate immune responses in children with ASD. To address this question in adults with high-functioning ASD (hf-ASD), we sought to investigate the role of natural killer (NK) cells in the persistence of ASD.MethodsNK cells from 35 adults with hf-ASD were compared to that of 35 healthy controls (HC), selected for the absence of any immune dysfunctions, at different time-points, and over a 2-year follow-up period for four patients. The phenotype and polyfunctional capacities of NK cells were explored according to infectious stigma and clinical parameters (IQ, social, and communication scores).ResultsAs compared to HC, NK cells from patients with hf-ASD showed a high level of cell activation (p < 0.0001), spontaneous degranulation (p < 0.0001), and interferon-gamma production (p = 0.0004), whereas they were exhausted after in vitro stimulations (p = 0.0006). These data yielded a specific HLA-DR+KIR2DL1+NKG2C+ NK-cell signature. Significant overexpression of NKG2C in hf-ASD patients (p = 0.0005), indicative of viral infections, was inversely correlated with the NKp46 receptor level (r = − 0.67; p < 0.0001), regardless of the IgG status of tested pathogens. Multivariate linear regression analysis also revealed that expression of the late-activating HLA-DR marker was both associated with structural language (r = 0.48; p = 0.007) and social awareness (r = 0.60; p = 0.0007) scores in adult patients with hf-ASD, while KIR2DL1 expression correlated with IQ scores (p = 0.0083).ConclusionsThis study demonstrates that adults with hf-ASD have specific NK-cell profile. Presence of NKG2C overexpression together with high-level activation of NK cells suggest an association with underlying pathogens, a hypothesis warranting further exploration in future studies.

FcγRIIIA-mediated activation of NK cells by IgG heavy chain complexed with MHC class II molecules.

Natural killer (NK) cells are a major FcγRIIIA-expressing lymphocyte population that mediate antibody-dependent cellular cytotoxicity. Although NK cells are critical for immunity against viruses and tumors, they are also activated in the joints of patients with rheumatoid arthritis (RA) and may be involved in disease progression. We previously found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins, such as IgG heavy chain (IgGH), to the cell surface via association with their peptide-binding grooves. Furthermore, we found that IgGHs bound to HLA class II molecules encoded by RA susceptibility alleles are specific targets for rheumatoid factor, an auto-antibody involved in RA. Here, we report that IgGHs bound to HLA class II molecules preferentially stimulate FcγRIIIA-expressing but not FcγRI-expressing cells. A significant correlation was observed between the reactivity of FcγRIIIA-expressing cells to IgGH complexed with a specific HLA-DR allele and the odds ratio for HLA-DR allele's association with RA. Moreover, primary human NK cells expressing FcγRIIIA demonstrated IFN-γ production and cytotoxicity against cells expressing IgGH complexed with HLA class II molecules. Our findings suggest that IgGH complexed with HLA class II molecules are involved in the activation of FcγRIIIA-expressing NK cells observed within arthritic joints.

P164 Repetitive in-vitro exposure to DFP affects MHC class I and class II antigen expression

O,O-Diisopropyl phosphofluoridate also known as Diisopropylfluorophosphate (DFP) a surrogate compound of nerve agents (Cyclosarin, VX, Sarin), is an organophosphate, used in this study to understand the mechanisms of toxicity on T cells, B cells and monocytes. Our aim was to study MHC class I & II antigen expression on T cells, B cells and monocytes after repetitive exposure to sub-lethal doses of DFP in-vitro. This study was conducted as a proof of concept since this compound was used in rodent models to study Gulf War Illness (GWI), which is a chronic multisymptom disorder affecting military veterans and civilian worker that served during 1990–91 Gulf War. We isolated T cells, B cells, and macrophages from 10 healthy donors. Freshly isolated cells were maintained in culture and were exposed to 0.1–100 μM DFP for 3 days at a 24 h interval. HLA typing was performed both by using the PCR-sequence specific primer (SSP) and by serological methods before exposure to DFP. After exposure, the cells were screened for the presence of the specific HLA-B and HLA-DR antigen. We isolated the culture supernatant to study the soluble HLA antigens. HLA typing was done for both A and B allele for T cells and DR for B cells using serology and by PCR-SSP. Once the antigens were delineated, after exposure they were tested for the specific antigen. Doses above 50 μM of DFP showed weak antigen expression. The cell surface antigen appeared weak by serology. The cell supernatant tested positive for HLA antigens, although they were combined with other proteins, it will be interesting to see how these HLA cell surface markers detach from the cell. We have isolated the supernatant for LC–MS analysis. Control cells that were not treated with DFP gave a consistently positive result and the HLA A, B, and DR were same as pre-exposure. There was no detectable sHLA in the supernatant. Exposure to organophosphate-based pesticides can cause immunotoxicity. Although we suspected and were trying to understand the GWI, these results offer a valid clue that there is relevance to HLA antigen expression and to look for soluble HLA antigen in the serum/plasma, as this sHLA, may very well initiate an autoimmune response by triggering inflammatory signals.

OR42 Reactivity of monoclonal antibodies for the detection of HLA class II surface expression: Mixing apples with oranges?

Aim The flow cytometry crossmatch (FCXM) is a widely used method to assess pre-transplant immunologic risk. One variable that may affect FCXM reactivity is variability in lymphocyte HLA surface expression. Therefore, assessing HLA density on donor cells in FCXM can help guide FCXM interpretation, particularly HLA Class II, due to the inherent high background reactivity of B cells. Using single antigen beads (SAB), we defined the reactivity of two commercially available monoclonal antibodies (moAb) that claim pan-reactivity to all HLA class II. Methods Mouse anti-human HLA class II clones WR18 and Tu39 were tested by Luminex SAB from two vendors. PE-labeled goat anti-mouse IgG was used as a detection antibody for both clones. Multiple dilutions of the moAb were tested to ensure bead saturation. Patterns of reactivity across antigens of different HLA loci were compared. Results Whereas WR18 bound to all HLA-DR antigens, Tu39 showed lower reactivity to DR7, DR12, and specific alleles of DR9 (DRB1∗09:01), DR14 (DRB1∗14:01, 14:04 and 14:54) and DR52 (DRB3∗01:01 and 03:01) [Fig. 1A, B]. This pattern aligned with a lack of reactivity to a specific epitope 60Y on these HLA alleles. Although reactivity varied, WR18 detected all HLA-DQ antigens. In contrast, Tu39 only bound to DQ2, DQ4 and a specific allele of DQ6 (DQB1∗06:01) [Fig. 1C, D]. HLA-DP was detected by both clones with similar broad reactivity to all antigens. Conclusions In contrast to WR18, Tu39 lacks reactivities to specific HLA-DR and -DQ antigens. This limited reactivity would affect assessment of HLA expression in FCXM when donor HLA types include antigens to which Tu39 is weakly or not reactive. Detailed knowledge of the specific reactivities of commercially available moAb is imperative for their usage in FCXM when analysis of HLA expression is part of the assessment. This information may also be valuable to the broader scientific community that may not have the technology readily available to investigate HLA-specific reactivity of moAb.

Genetic variants in the HLA class II region associated with risk of cutaneous squamous cell carcinoma.

The immune system has been implicated in the pathophysiology of cutaneous squamous cell carcinoma (cSCC) as evidenced by the substantially increased risk of cSCC in immunosuppressed individuals. Associations between cSCC risk and single nucleotide polymorphisms (SNPs) in the HLA region have been identified by genome-wide association studies (GWAS). The translation of the associated HLA SNPs to structural amino acids changes in HLA molecules has not been previously elucidated. Using data from a GWAS that included 7238 cSCC cases and 56,961 controls of non-Hispanic white ancestry, we imputed classical alleles and corresponding amino acid changes in HLA genes. Logistic regression models were used to examine associations between cSCC risk and genotyped or imputed SNPs, classical HLA alleles, and amino acid changes. Among the genotyped SNPs, cSCC risk was associated with rs28535317 (OR = 1.20, p = 9.88 × 10- 11) corresponding to an amino-acid change from phenylalanine to leucine at codon 26 of HLA-DRB1 (OR = 1.17, p = 2.48 × 10- 10). An additional independent association was observed for a threonine to isoleucine change at codon 107 of HLA-DQA1 (OR = 1.14, p = 2.34 × 10- 9). Among the classical HLA alleles, cSCC was associated with DRB1*01 (OR = 1.18, p = 5.86 × 10- 10). Conditional analyses revealed additional independent cSCC associations with DQA1*05:01 and DQA1*05:05. Extended haplotype analysis was used to complement the imputed haplotypes, which identified three extended haplotypes in the HLA-DR and HLA-DQ regions. Associations with specific HLA-DR and -DQ alleles are likely to explain previously observed GWAS signals in the HLA region associated with cSCC risk.

Assessing antigen specific HLA-DR+ antibody secreting cell (DR+ASC) responses in whole blood in enteric infections using an ELISPOT technique

Antibody secreting cells (ASCs) generate antibodies in an antigen-specific manner as part of the adaptive immune response to infections, and these cells increase their surface expression of HLA-DR. We have studied this parameter (HLA-DR+ ASC) in patients with recent diarrheal infection using immuno-magnetic cell sorting and an enzyme linked immunospot (ELISPOT) technique that requires only one milliliter of blood. We validated this approach in adult patients with cholera (n = 15) or ETEC diarrhea (n = 30) on days 2, 7 and 30 after showing clinical symptom at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) hospital in Dhaka, and we compared responses to age-matched healthy controls (n = 7). We found that HLA-DR+ ASC (DR+ASC) responses specific both for T cell-dependent (cholera toxin B subunit), and T cell-independent (lipopolysaccharide) antigens were elevated at day 7 after showing clinical cholera symptom. Similarly, DR+ASCs were elevated against both heat-labile toxin and colonization factors following ETEC infection. We observed significant correlations between antigen-specific DR+ASC responses and antigen-specific, gut homing ASC and plasma antibody responses. This study demonstrates that a simple ELISPOT procedure allows determination of antigen-specific ASC responses using a small volume of whole blood following diarrhea. This technique may be particularly useful in studying DR+ASC responses in young children and infants, either following infection or vaccination.

Molecular mimicry and autoimmune thyroid disease.

Hypothesized 40years ago, molecular mimicry has been thereafter demonstrated as an extremely common mechanism by which microbes elude immune response and modulate biosynthetic/metabolic pathways of the host. In genetically predisposed persons and under particular conditions, molecular mimicry between microbial and human antigens can turn a defensive immune response into autoimmunity. Such triggering role and its pathogenetic importance have been investigated and demonstrated for many autoimmune diseases. However, this is not the case for autoimmune thyroid disease, which appears relatively neglected by this field of research. Here we review the available literature on the possible role of molecular mimicry as a trigger of autoimmune thyroid disease. Additionally, we present the results of in silico search for amino acid sequence homologies between some microbial proteins and thyroid autoantigens, and the potential pathogenetic relevance of such homologies. Relevance stems from the overlap with known autoepitopes and the occurrence of specific HLA-DR binding motifs. Bioinformatics data published by our group support and explain the triggering role of Borrelia, Yersinia, Clostridium botulinum, Rickettsia prowazekii and Helicobacter pylori. Our new data suggest the potential pathogenic importance of Toxoplasma gondii, some Bifidobacteria and Lactobacilli, Candida albicans, Treponema pallidum and hepatitis C virus in autoimmune thyroid disease, indicating specific molecular targets for future research. Additionally, the consistency between in silico prediction of cross-reactivity and experimental results shows the reliability and usefulness of bioinformatics tools to precisely identify candidate molecules for in vitro and/or in vivo experiments, or at least narrow down their number.

Langerhans Cell Histiocytosis Shows Distinct Cytoplasmic Expression of Major Histocompatibility Class II Antigens.

Langerhans cell histiocytosis (LCH) is a monoclonal proliferation of antigen presenting cells (APC). In benign APCs, antigen loading occurs in the Major Histocompatibility class II (MHCII)-lysosomal compartment of the endocytic pathway followed by transport to the cell surface upon antigen stimulation. The pattern of MHC II expression in LCH is not well characterized. The cellular localization of MHCII was determined using immunohistochemisty (IHC). Staining pattern for the representative MHCII molecule, HLA-DR, (cell surface, cytoplasmic granular, or cytoplasmic globular) and intensity (0 to 3+) were recorded for normal tissues and 44 LCH samples along with available clinicopathologic features. Results were confirmed with a different antibody to confirm the appearance. In the normal tissue survey, strong HLA-DR cell surface expression was present on APCs, benign B cells, some T cells, and pulmonary macrophages. A granular cytoplasmic staining pattern (without surface expression) was seen in benign Langerhans cells (LCs) in the skin and histiocytes. Strikingly, all 44 LCH samples demonstrated both cytoplasmic granular and an unusual "globular" staining pattern with no surface staining. This is the first report of a highly specific HLA-DR staining pattern in LCH detected by IHC. The cytoplasmic perinuclear globular localization of MHCII may possibly be useful in diagnostics and may result from an immature/antigen-naïve differentiation state of the neoplastic cell.

CD4⁺ T cells in chronic autoantigenic stimulation in MGUS, multiple myeloma and Waldenström's macroglobulinemia.

Hyperphosphorylated paratarg-7 (pP-7) carrier state is the strongest and most frequent molecular risk factor for MGUS, multiple myeloma (MM) and Waldenström's macroglobulinemia (WM), inherited autosomal-dominantly and, depending on the ethnic background, found in up to one third of patients with MGUS/MM. Since P-7 is the antigenic target of paraproteins that do not distinguish between wtP-7 and pP-7, we investigated CD4(+) T-cell responses in pP-7(+) patients and controls. Peptides spanning amino acids 1-35 or 4-31 containing phosphorylated or nonphosphorylated serine17 were used for stimulation. CD4(+) cells from 9/14 patients (65%) showed a pP-7 specific HLA-DR restricted response. These results demonstrate that pP-7 specific CD4(+) cells can mediate help for pP-7 specific chronic antigenic stimulation of P-7 specific B cells, which might ultimately result in the clonal evolution of a B cell into MGUS/MM/WM producing a P-7 specific paraprotein. Prerequisites for pP-7 specific stimulation of CD4(+) cells appear to be both a pP-7 carrier state and an HLA-DR subtype able to present and recognize pP-7. Our results serve as an explanation for the exclusive autoimmunogenicity of the hyperphosphorylated variant of P-7 and for the different hazard ratios of pP-7 carriers from different ethnic origins to develop MGUS/MM/WM.

Factor VIII Proteins Having a Rationally Modified, Immunodominant T-Cell Epitope Demonstrate Normal Procoagulant Activity, Bind To VWF With High Affinity, and Are Markedly Less Stimulatory To FVIII-Specific Human T Cells

Neutralizing anti-factor VIII (FVIII) antibodies, referred to clinically as “inhibitors”, can develop as an alloimmune response in hemophilia A patients receiving FVIII infusions as replacement therapy. Immune Tolerance Induction, consisting of intensive FVIII treatment in an effort to tolerize patients to FVIII, is extraordinarily expensive and not always successful. New approaches to avoid inhibitor development, and to treat patients who develop this deleterious immune response, are needed. Initial stages of inhibitor development include FVIII uptake and processing into peptides by antigen-presenting cells such as dendritic cells (DCs), presentation of FVIII peptides on MHC Class II (HLA) receptors on the cell surface, and recognition of HLA-peptide complexes by one or more circulating T cells. Subsequent signaling through immunological synapses between antigen-presenting cells and T-cell receptors causes proliferation of effector T cells, which secrete cytokines promoting anti-FVIII antibody production.Our laboratory has been identifying T-cell epitopes in FVIII, which are amino acid sequences 11-15 residues long that bind to specific HLA-DR proteins and are in turn recognized by T cells. FVIII 2194-2205 comprises an immunodominant T-cell epitope that binds to HLA-DRB1*01:01. Peptide-MHC binding experiments established that the side chains of amino acid residues F2196, M2199, A2201 and S2204 fit into the HLA-DRB1*01:01 peptide-binding groove, allowing peptides containing this sequence to be presented on the surface of antigen-presenting cells. Fluorescent HLA-DRB1*01:01 tetramers loaded with FVIII2194-2213 were used to stain and isolate FVIII-specific T cells from 3 hemophilia A subjects who had an HLA-DRB1*01:01 allele. These cells were expanded in culture to generate T-cell clones and polyclonal lines that recognize this sequence and proliferate in response to it. Stimulation of clones with FVIII peptides containing systematic alanine substitutions demonstrated that MHC anchor residues F2196 and M2199 are important for the immunogenicity of this T-cell epitope. The clones were then stimulated with FVIII peptides and recombinant FVIII-C2 domain proteins in which F2196 was changed to a series of other residues. The effect of substitutions at M2199 was examined using the ProPred computer prediction program. Three substitutions that significantly reduced T-cell proliferation (F2196A, F2196L, F2196K) or were predicted to do so (M2199A, M2199W, M2199R) were introduced into recombinant, B-domain-deleted (BDD)-FVIII. The present study tests the hypothesis that less immunogenic FVIII proteins having normal FVIII procoagulant activity can be produced through rational modification of T-cell epitopes. The F2196K and M2199A muteins were expressed at levels similar to wild-type (WT)-BDD-FVIII in BHK-M cells and were purified from serum-free BHK-M cell supernatants. Purified CD14-positive monocytes from individuals with the HLA-DRB1*01:01 allele were differentiated into DCs and used for antigen-presentation assays in which 4 hemophilic T-cell clones and 2 polyclonal lines were added to the DCs and stimulated with FVIII2194-2213, WT-FVIII, WT-BDD-FVIII and the BDD-FVIII muteins. One clone proliferated weakly in response to both the WT and mutant proteins, while the remaining 3 clones and 2 lines showed markedly less proliferation in response to the BDD-FVIII muteins. BDD-FVIII-2196K and 2199A had specific activities similar to that of WT-BDD-FVIII (by chromogenic and clotting assays), and surface plasmon resonance confirmed that they retained high-affinity binding to von Willebrand factor. These results provide proof-of-principle for the design of less immunogenic FVIII proteins targeted to specific subsets of hemophilia A patients. Disclosures:Pratt:Bayer, Pfizer, CSL Behring: Research Funding; Puget Sound Blood Center Research Institute: sequence-modified FVIII variants Patents & Royalties.

SP0177 Lyme disease – old and new insights

Lyme disease was recognized as a separate entity in 1976 because of geographic clustering of children in Lyme, Connecticut who were thought to have juvenile rheumatoid arthritis, a presumed autoimmune...

A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.