Human HLA-DR Research Articles

A Recombinant Multivalent Vaccine (rCpa1) Induces Protection for C57BL/6 and HLA Transgenic Mice against Pulmonary Infection with Both Species of Coccidioides.

Coccidioidomycosis is caused by Coccidioides posadasii (Cp) and Coccidioides immitis (Ci), which have a 4-5% difference in their genomic sequences. There is an urgent need to develop a human vaccine against both species. A previously created recombinant antigen (rCpa1) that contains multiple peptides derived from Cp isolate C735 is protective against the autologous isolate. The focus of this study is to evaluate cross-protective efficacy and immune correlates by the rCpa1-based vaccine against both species of Coccidioides. DNA sequence analyses of the homologous genes for the rCpa1 antigen were conducted for 39 and 17 clinical isolates of Cp and Ci, respectively. Protective efficacy and vaccine-induced immunity were evaluated for both C57BL/6 and human HLA-DR4 transgenic mice against five highly virulent isolates of Cp and Ci. There are total of seven amino acid substitutions in the rCpa1 antigen between Cp and Ci. Both C57BL/6 and HLA-DR4 mice that were vaccinated with an rCpa1 vaccine had a significant reduction of fungal burden and increased numbers of IFN-γ- and IL-17-producing CD4+ T cells in the first 2 weeks post challenge. These data suggest that rCpa1 has cross-protection activity against Cp and Ci pulmonary infection through activation of early Th1 and Th17 responses.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Human HLA-DR+CD27+ regulatory T cells show enhanced antigen-specific suppressive function.

Regulatory T cells (Tregs) have potential for the treatment of autoimmune diseases and graft rejection. Antigen specificity and functional stability are considered critical for their therapeutic efficacy. In this study, expansion of human Tregs in the presence of porcine PBMCs (xenoantigen-expanded Tregs, Xn-Treg) allowed the selection of a distinct Treg subset, coexpressing the activation/memory surface markers HLA-DR and CD27 with enhanced proportion of FOXP3+Helios+ Tregs. Compared with their unsorted and HLA-DR+CD27+ double-positive (DP) cell-depleted Xn-Treg counterparts, HLA-DR+CD27+ DP-enriched Xn-Tregs expressed upregulated Treg function markers CD95 and ICOS with enhanced suppression of xenogeneic but not polyclonal mixed lymphocyte reaction. They also had less Treg-specific demethylation in the region of FOXP3 and were more resistant to conversion to effector cells under inflammatory conditions. Adoptive transfer of porcine islet recipient NOD/SCID IL2 receptor γ-/- mice with HLA-DR+CD27+ DP-enriched Xn-Tregs in a humanized mouse model inhibited porcine islet graft rejection mediated by 25-fold more human effector cells. The prolonged graft survival was associated with enhanced accumulation of FOXP3+ Tregs and upregulated expression of Treg functional genes, IL10 and cytotoxic T lymphocyte antigen 4, but downregulated expression of effector Th1, Th2, and Th17 cytokine genes, within surviving grafts. Collectively, human HLA-DR+CD27+ DP-enriched Xn-Tregs expressed a specific regulatory signature that enabled identification and isolation of antigen-specific and functionally stable Tregs with potential as a Treg-based therapy.

Risk factors and outcomes of IgA nephropathy recurrence after kidney transplantation: a systematic review and meta-analysis.

IgA nephropathy may recur in patients receiving kidney transplantation due to IgA nephropathy induced renal failure. The risk factors for recurrence are still at issue. The aim of this study was to conduct a systematic review and meta-analysis to assess risk factors and outcomes for IgA nephropathy recurrence. We used PubMed, EMBASE, Cochrane Library, Web of Science, Scopus, CNKI, WanFang, VIP and CBM to search for relevant studies published in English and Chinese. Cohort or case-control studies reporting risk factors or outcomes for IgA nephropathy recurrence were included. Fifty-eight studies were included. Compare to no recurrence group, those with IgAN recurrence had younger age (mean difference [MD]=-4.27 years; risk ratio [RR]=0.96), younger donor age (MD=-2.19 years), shorter time from IgA nephropathy diagnosis to end stage renal disease (MD=-1.84 years; RR=0.94), shorter time on dialysis (MD=-3.14 months), lower human leukocyte-antigen (HLA) mismatches (MD=-0.11) and HLA-DR mismatches (MD=-0.13). HLA-B46 antigen (RR=0.39), anti-IL-2-R antibodies induction (RR=0.68), mycophenolate mofetil (RR=0.69), and pretransplant tonsillectomy (RR=0.43) were associated with less IgAN recurrence. Of note, male recipient gender (RR=1.17), related donor (RR=1.53), retransplantation (RR=1.43), hemodialysis (RR=1.68), no induction therapy (RR=1.73), mTOR inhibitor (RR=1.51), angiotensin-converting enzyme inhibitors or angiotensin-receptor blockers (RR=1.63) were risk factors for IgAN recurrence. Recurrence increased the risk of graft loss (RR=2.19). This study summarized the risk factors for recurrence of IgA nephropathy after kidney transplantation. Well-designed prospective studies are warranted for validation. https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=377480, identifier CRD42022377480.

Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and β chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR β chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.

Five commercially-available antibodies react differentially with allelic forms of human HLA-DR beta chain

Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population.

Tooth Root Formation

The formation of the tooth crown implies dentin and pulp construction (including mantle dentin, odontoblasts, Höhl cells, and vascular, axons and pulp cells). The root formation involves acellular and cellular cementum, peripheral and central dentins. Vascular cells and nerves are located in the central part of the root, whereas the superficial layer (Hopewell-Smith and Tomes granular layers) are located at the periphery. In the central part of the pulp, fibroblasts and immunocompetent cells are identified as T-lymphocytes, B-lymphocytes, dendritic cells, NK cells. In human deciduous teeth, HLA-DR+, CD68+, and factor XIIIa are present, whereas T and B-lymphocytes form clusters. The Hertwig’s Epithelial Enamel Epithelium (HERS’s) is formed by two layers. The HERS’s cells dissociate, and cells, taking origin from the dental follicular sac and from the papilla, migrates. The OEE becomes periodontal ligament cells whereas the IEE becomes cementoblasts. The interconversion of epithelial cells into mesenchymal cells seems to be a crucial event in tooth root formation. Tooth eruption is associated to root lengthening. The temporary tooth is gradually destroyed (root cementoclasts), whereas the permanent coronal part of the tooth erupts and acquire it final shape and position.

Combinatorial in silico and in vivo evaluation of immune response elicitation by the affibody ZHER2

Due to the high affinity for binding to target molecules and also other unique attributes, affibodies have a great potential to be used in immunotherapeutic and diagnostic approaches. However, the possibility of undesirable immune response is still a great concern. In the current study, we investigated the possible antigenicity, allergenicity and cytotoxicity of the HER2-targeting affibody ZHER2. The binding affinity of potential epitopes of the affibody to murine major histocompatibility complex (MHC) molecules was investigated by immunoinformatics tools and docking approaches. The possible interaction of ZHER2 with human leukocyte antigens HLA-DP, HLA-DM, HLA-DQ and HLA-DR was also studied by protein-protein docking. Additionally, the synthesized affibody gene was expressed and the protein was purified for boosted immunization of Balb/c mice. Induced secretion of IFN-γ, IL-2, IL-4 and IL-10, and total serum IgG were assessed in the immunized mice. Furthermore, MTT cell viability test was performed to evaluate the cytotoxicity of ZHER2 in splenocytes of the treated mice. In silico analyses showed the possible induction of the immune response by ZHER2. While the affibody could elicit the secretion of cellular immune cytokines, it could not induce a significant humoral response in the treated mice and did not show any cytotoxic effects on the exposed splenocytes. These findings explain the practicability of ZHER2 for therapeutic and in vivo diagnostic usages, though its ubiquitous application may need more studies.

CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration.

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Plasmodium falciparum Blood Stage Antimalarial Vaccines: An Analysis of Ongoing Clinical Trials and New Perspectives Related to Synthetic Vaccines.

Plasmodium falciparum malaria is a disease causing high morbidity and mortality rates worldwide, mainly in sub-Saharan Africa. Candidates have been identified for vaccines targeting the parasite’s blood stage; this stage is important in the development of symptoms and clinical complications. However, no vaccine that can directly affect morbidity and mortality rates is currently available. This review analyzes the formulation, methodological design, and results of active clinical trials for merozoite-stage vaccines, regarding their safety profile, immunological response (phase Ia/Ib), and protective efficacy levels (phase II). Most vaccine candidates are in phase I trials and have had an acceptable safety profile. GMZ2 has made the greatest progress in clinical trials; its efficacy has been 14% in children aged less than 5 years in a phase IIb trial. Most of the available candidates that have shown strong immunogenicity and that have been tested for their protective efficacy have provided good results when challenged with a homologous parasite strain; however, their efficacy has dropped when they have been exposed to a heterologous strain. In view of these vaccines’ unpromising results, an alternative approach for selecting new candidates is needed; such line of work should be focused on how to increase an immune response induced against the highly conserved (i.e., common to all strains), functionally relevant, protein regions that the parasite uses to invade target cells. Despite binding regions tending to be conserved, they are usually poorly antigenic and/or immunogenic, being frequently discarded as vaccine candidates when the conventional immunological approach is followed. The Fundación Instituto de Inmunología de Colombia (FIDIC) has developed a logical and rational methodology based on including conserved high-activity binding peptides (cHABPs) from the main P. falciparum biologically functional proteins involved in red blood cell (RBC) invasion. Once appropriately modified (mHABPs), these minimal, subunit-based, chemically synthesized peptides can be used in a system covering the human immune system’s main genetic variables (the human leukocyte antigen HLA-DR isotype) inducing a suitable, immunogenic, and protective immune response in most of the world’s populations.

Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction

BackgroundSmoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction.ResultsThis paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2- to 19-fold enhanced catalytic activity compared to wt at 10 μM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen’s access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity.ConclusionsSystematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme’s immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

Entry of the bat influenza H17N10 virus into mammalian cells is enabled by the MHC class II HLA-DR receptor.

Haemagglutinin and neuraminidase surface glycoproteins of the bat influenza H17N10 virus neither bind to nor cleave sialic acid receptors, indicating that this virus employs cell entry mechanisms distinct from those of classical influenza A viruses. We observed that certain human haematopoietic cancer cell lines and canine MDCK II cells are susceptible to H17-pseudotyped viruses. We identified the human HLA-DR receptor as an entry mediator for H17 pseudotypes, suggesting that H17N10 possesses zoonotic potential.

Abstract 4445: MHC class II antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides

Abstract A key goal in immuno-oncology is the identification of tumor antigens recognized by T cells. Significant progress has been made in predicting MHC class I presentation of tumor-specific antigens (peptides) recognized by CD8 reactive T cells. However, predicting antigens recognized by CD4 T cells that are presented by the MHC class II has proven to be more challenging. Studies have shown binding affinity to be less predictive for MHC class II presentation than for class I. Class II antigen-directed therapeutics require accurate antigen identification from patient samples, which remains elusive today. Methods: We focused initially on antigen presentation by the HLA-DR and generated a dataset of human tumor transcriptomes and HLA-DR immunopeptidomes from resections of B cell lymphomas (N=39). Transcriptomes were obtained by NGS of exome-captured cDNA and immunopeptidomes by immunoprecipitation using the HLA-DR specific Ab L243 and MS/MS. Each sample was typed for HLA-DRB1,3,4,5 using standard methods. Additionally, we obtained published class II mass spectrometry data for two B cell lines, each of which expressed a single common HLA class II allele (HLA-DRB1*15:01 and HLA-DRB5*01:01). RNA sequencing data was not available for either cell line; therefore, we substituted RNA-sequencing data from a different B cell line, B721.221. We combined these data to train a deep learning model of Class II HLA peptide presentation. Our model addressed two key challenges: (1) learning HLA-allele-specific models from tumor and normal data where each sample expressed up to 4 unique HLA-DR alleles and (2) reflecting information about all aspects of HLA presentation, including gene expression, antigen processing and stable binding of peptides to HLA. We evaluated the performance of the model on two independent test datasets. First, we tested the model on HLA peptides from a held-out sample from the lymphoma training dataset. Second, we tested the model on a separate public cell line expressing both HLA-DRB1*15:01 and HLA-DRB5*01:01. Results: An average of 567 training and 203 testing peptides at q<0.01 and >50M unique transcriptome reads were obtained from the lymphoma samples. From cell-lines, 433 peptides for training and 223 peptides for testing were used. The model demonstrated a significant improvement in prediction accuracy, achieving >10%-point gain in area under the ROC curve (ROC AUC) vs a standard binding affinity-based predictor. On the held-out lymphoma test data, it achieved a ROC AUC of .95 vs the binding affinity model ROC AUC .80. On the cell-line test data, it achieved a ROC AUC of .90 vs the binding affinity model ROC AUC of .79. Conclusion: We used a large dataset of transcriptomes and HLA peptidomes to train a deep learning model for HLA class II antigen presentation. The new model significantly outperforms standard methods and advances in silico HLA class II antigen selection for personalized cancer immunotherapy. Citation Format: Tommy Boucher, Matthew Davis, Christine Palmer, Tyler Murphy, Andrew Clark, Fujiko Duke, Aaron Yang, Lauren Young, Karin Jooss, Mojca Skoberne, Josh Francis, Roman Yelensky, James Sun, Jennifer Busby. MHC class II antigen identification for cancer immunotherapy by deep learning on tumor HLA peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4445.

Role of Dendrtic Cells in IgA Nephropathy Pathogenesis

Objective to discuss the possible role and mechanism of DCs in IgAN attack. Method Stimulating factors such as recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL-4) and tumor necrosis factor-a (TNF-a) etc. were used in vitro jointly to induce and culture DCs, a flow cytometry was used to detect expression of HLA-DR, CD83 and CDla of DCs membrane surface molecules, the MTT method was used to detect capacity of DCs of the IgAN patient group to stimulate proliferation of allogeneic T cells and the difference between the levels of interleukin 6 (IL-6) and interleukin 12 (IL-12) secreted by DCs and that of the normal control group. Result Combined application of cytokines GM-CSF, IL-4 and TNF-a is able to induce proliferation and differentiation of peripheral blood mononuclear cell into a mature dendritic cell. The surface of mature dendritic cells highly expresses human leucocyte antigen HLA-DR and surface maturity markers of relative specificity of dendritic cells, CD83 and CD1a. The capacity of DCs of the IgAN patient group to stimulate allogeneic T lymphocyte proliferation is higher than that of the normal control group and the difference has statistical significance (P<0.05). The capacity of DCs of the patient group influenced by lipopolysaccharide (LPS) to stimulate allogeneic T lymphocyte proliferation significantly increases compared with that of DCs of the patient group not influenced by lipopolysaccharide (LPS) and the difference has statistical significance (P<0.05). The IL-6 secreted by the DCs of the IgAN patient group is higher than that of the normal control group and the difference between the two groups has statistical significance (P<0.05). The IL-12 secreted by the DCs of the IgAN patient group is lower than that of the normal control group and the difference between the two groups has statistical significance (P<0.05). Conclusion DCs may regulate the balance between Thl/Th2 cells by secreting cytokines so as to play an imporat role in occurence and progression of IgAN and such factors as infection etc. may strengthen the functions of DCs thus easily triggering IgAN.

Glucan-Chitin Particles Enhance Th17 Response and Improve Protective Efficacy of a Multivalent Antigen (rCpa1) against Pulmonary Coccidioides posadasii Infection.

Developing an effective and safe recombinant vaccine requires microbe-specific antigens combined with an adjuvant/delivery system to strengthen protective immunity. In this study, we designed and expressed a multivalent recombinant Coccidioides polypeptide antigen (rCpa1) that consists of three previously identified antigens (i.e., Ag2/Pra, Cs-Ag, and Pmp1) and five pathogen-derived peptides with high affinity for human major histocompatibility complex class II (MHC-II) molecules. The purified rCpa1 was encapsulated into four types of yeast cell wall particles containing β-glucan, mannan, and chitin in various proportions or was mixed with an oligonucleotide (ODN) containing two methylated dinucleotide CpG motifs. This multivalent antigen encapsulated into glucan-chitin particles (GCP-rCpa1) showed significantly greater reduction of fungal burden for human HLA-DR4 transgenic mice than the other adjuvant-rCpa1 formulations tested. Among the adjuvants tested, both GCPs and β-glucan particles (GPs) were capable of stimulating a mixed Th1 and Th17 response. Mice vaccinated with GCP-rCpa1 showed higher levels of interleukin 17 (IL-17) production in T-cell recall assays and earlier lung infiltration by activated Th1 and Th17 cells than GP-rCpa1-vaccinated mice. Both C57BL/6 and HLA-DR4 transgenic mice that were vaccinated with the GCP-rCpa1 vaccine showed higher survival rates than mice that received GCPs alone. Concurrently, the GCP-rCpa1 vaccine stimulated greater infiltration of the injection sites by macrophages, which engulf and process the vaccine for antigen presentation, than the GP-rCpa1 vaccine. This is the first attempt to systematically characterize the presentation of a multivalent coccidioidomycosis vaccine encapsulated with selected adjuvants that enhance the protective cellular immune response to infection.

Proinsulin-mediated induction of type 1 diabetes in HLA-DR4-transgenic mice

Antigen-specific immunotherapy of autoimmune disease currently remains the only potentially curative approach. However, translation of promising pre-clinical results into successful clinical application has proven challenging. In part, this is because pre-clinical findings in mouse models have to be redesigned for human application due to differences in MHC II. To reduce the gap between pre-clinical and clinical studies, we have created a novel mouse model that expresses human HLA-DR4, but no endogenous MHC on antigen-presenting cells. Moreover, human B7.1 (CD80) is expressed in the pancreatic islets under the control of the rat insulin promoter. Although this model does not develop diabetes spontaneously, it is susceptible to the induction of type 1 diabetes by challenging mice with overlapping peptides derived from murine proinsulin-2 in adjuvant. Unlike the NOD model of spontaneous type 1 diabetes, but akin to the human condition, this model does not have a gender bias. Furthermore, similar to the human condition, the disease is characterised by a diverse leucocyte infiltration of the pancreatic islets and the formation of anti-proinsulin auto-antibodies. The model that we report here offers detailed insights into type-1 diabetes and is expected to prove instrumental when studying the mechanism of action in translational, antigen-specific immunotherapy.

Abstract 5616: Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients

Abstract For many years, the clinical responses observed in cancer patients treated with antitumor antibodies have been viewed only as a consequence of the recruitment and activation of innate immune cells. Several preclinical studies have indicated that anti-tumor antibodies can also induce a potent anti-tumor adaptive T-cell response capable of protecting animals from tumor challenge. Our studies have shown that CD4+ Th1 cells play a central role in the response to anti-CD20 tumor therapy by opposing Treg expansion and that CD20-specific memory T cells are responsible for anti-tumor protection observed in surviving animals subjected to tumor rechallenge. Whether an adaptive T-cell response is elicited in patients with Follicular Lymphoma (FL) receiving anti-CD20-based treatment is not known. In the present work, we analyzed peripheral T cells before and during anti-CD20-chemotherapy treatment of FL patients (R-CHOP) with high tumor burden. We first examined in ELISPOT assays the IFN-γ production by PBMC from 20 patients against human CD20-derived peptides previously selected by combining in silico and in vivo screening using transgenic human HLA-DR mice. Both FL patients before treatment and patients undergoing R-CHOP treatment exhibited peripheral T-cell responses to the selected CD20-derived peptides. However, these responses were markedly lower than those observed in healthy donors. Similar results were also obtained when T cell responses against viruses commonly infecting large numbers of individuals (CMV, EBV, influenza) were assessed, suggesting that high tumor burden FL patients exhibit an immune exhaustion of T cells. Multiparametric flow cytometry analyses suggested that their poor ability to respond to viral and CD20-derived peptides is related to the presence of an immunocompromised and exhausted blood T-cell peripheral compartment with higher percentages of TIGIT- and PD-1-expressing T cells, of peripheral Treg, and terminally differentiated CD8+ TEMRA. R-CHOP therapy induced a shift of CD4+ and CD8+ T cells toward a central memory phenotype (TCM), an additional shift of CD8+ T cells to a naive phenotype, and a decrease in the number of PD-1+ and TIGIT+ T cells, making these cells evolving toward a lesser degree of exhaustion. However, it did not restore a level of specific responses similar to those observed in healthy donors. These results support the use of anti-PD-1 and anti-TIGIT antibodies in combination with anti-CD20 in FL patients with high-tumor burden. Citation Format: Benoit Milcent, Pauline Brice, Catherine Thieblemont, Jean-Luc Teillaud, Sophie Siberil. Rituximab chemotherapy induces a partial recovery from T-cell exhaustion in follicular lymphoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5616.

A humanized HLA-DR4 mouse model for autoimmune myocarditis

Myocarditis, the principal cause of dilated cardiomyopathy and heart failure in young adults, is associated with autoimmunity to human cardiac α-myosin (hCAM) and the DR4 allele of human major histocompatibility II (MHCII). We developed an hCAM-induced myocarditis model in human HLA-DR4 transgenic mice that lack all mouse MHCII genes, demonstrating that immunization for 3weeks significantly increased splenic T-cell proliferative responses and titres of IgG1 and IgG2c antibodies, abolished weight gain, provoked cardiac inflammation and significantly impaired cardiac output and fractional shortening, by echocardiography, compared to adjuvant-injected mice. Neither cardiac dilatation nor fibrosis occurred at this time point but prolonging the experiment was associated with mortality. Treatment with mixtures of hCAM derived peptides predicted to have high affinity for DR4 significantly preserved ejection fraction and fractional shortening. Our new humanized mouse model of autoimmune cardiomyopathy should be useful to refine hCAM-derived peptide treatment.

Lack of Evidence for Molecular Mimicry in HIV-Infected Subjects.

Previous studies in HIV patients have reported autoantibodies to several human proteins, including erythropoietin (EPO), interferon-α (IFN-α), interleukin-2 (IL-2), and HLA-DR, as potential mediators of anemia or immunosuppression. The etiology of these autoantibodies has been attributed to molecular mimicry between HIV epitopes and self-proteins. Here, the Luciferase Immunoprecipitation System (LIPS) was used to investigate the presence of such autoantibodies in HIV-infected adults. High levels of antibodies to HIV proteins such as capsid (p24), matrix (p17), envelope (gp41), and reverse transcriptase (RT) were detected using LIPS in both untreated and anti-retroviral-treated HIV-infected individuals but not in uninfected controls. LIPS readily detected anti-EPO autoantibodies in serum samples from subjects with presumptive pure red cell aplasia but not in any of the samples from HIV-infected or uninfected individuals. Similarly, subjects with HIV lacked autoantibodies to IFN-α, IL-2, HLA-DR and the immunoglobulin lambda light chain; all purported targets of molecular mimicry. While molecular mimicry between pathogen proteins and self-proteins is a commonly proposed mechanism for autoantibody production, the findings presented here indicate such a process is not common in HIV disease.