CD8+ T Cells Expressing an HLA-DR1 Chimeric Antigen Receptor Target Autoimmune CD4+ T Cells in an Antigen-Specific Manner and Inhibit the Development of Autoimmune Arthritis.

Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed using DR1-restricted CD4+ T cells that recognize other Ags. When B6.DR1 mice, in which autoimmune arthritis had been induced, were treated with the DR1-CII CAR T cells, the CII-specific autoimmune CD4+ T cell response was significantly decreased, autoantibody production was suppressed, and the incidence and severity of the autoimmune arthritis was diminished. These data demonstrate that HLA-DR CAR T cells have the potential to provide a highly specific therapeutic approach for the treatment of autoimmune disease.

Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

The Thermodynamic Mechanism of Peptide-MHC Class II Complex Formation Is a Determinant of Susceptibility to HLA-DM.

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306-319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non-compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by "sensing" flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones.

Evaluating the Role of HLA-DM in MHC Class II-Peptide Association Reactions.

Ag presentation by MHC class II (MHC II) molecules to CD4(+) T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a nonclassical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of Ag presentation. In this study, we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association by measuring real-time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide-averse conformation of MHC II to an active peptide-receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during Ag presentation.

Uveitis in children.

PURPOSE OF REVIEW: The review provides updates on novel risk markers for the development of pediatric inflammatory uveitis and a severe disease course, on treatment of refractory disease, and on the measurement of visual outcomes. RECENT FINDINGS: There are several new genetic markers, biomarkers, and clinical factors that may influence a child's uveitis disease course. It is important to identify children at risk for poor visual outcomes and who are refractory to traditional therapy. Racial disparities have recently been reported. We describe agents of potential benefit. In addition, we discuss the importance of patient reported outcomes in this population. SUMMARY: Uveitis can lead to vision-threatening complications. Timely and aggressive treatment of children identified to be at risk for a severe uveitis course may lead to improved outcomes.

Vaccine-induced Aß-specific CD8+ T cells do not trigger autoimmune neuroinflammation in a murine model of Alzheimer's disease.

BACKGROUND: Active immunization against Aß was reported to have a therapeutic effect in murine models of Alzheimer's disease. Clinical Aß vaccination trial AN1792 was interrupted due to the development in 6 % of the patients of meningoencephalitis likely involving pro-inflammatory CD4(+) T cells. However, the potential implication of auto-aggressive anti-Aß CD8(+) T cells has been poorly investigated. METHODS: Potential MHC-I-restricted Aß-derived epitopes were first analyzed for their capacity to recruit functional CD8(+) T cell responses in mouse models. Their impact on migration of CD8(+) T cells into the brain parenchyma and potential induction of meningoencephalitis and/or neuronal damage was investigated upon vaccination in the APPPS1 mouse model of AD. RESULTS: We identified one nonamer peptide, Aß33-41, which was naturally processed and presented in association with H-2-D(b) molecule on neurons and CD11b(+) microglia. Upon optimization of anchor residues for enhanced binding to H-2-D(b), immunization with the modified Aß33-41NP peptide elicited Aß-specific IFNγ-secreting CD8(+) T cells, which are cytotoxic towards Aß-expressing targets. Whereas T cell infiltration in the brain of APPPS1 mice is dominated by CD3(+)CD8(-) T cells and increases with disease evolution between 4 and 7 months of age, a predominance of CD3(+)CD8(+) over CD3(+)CD8(-) cells was observed in 6- to 7-month-old APPPS1 but not in WT animals, only after vaccination with Aß33-41NP. The number of CD11b(+) mononuclear phagocytes, which significantly increases with age in the brain of APPPS1 mice, was reduced following immunization with Aß33-41NP. Despite peripheral activation of Aß-specific CD8(+) cytotoxic effectors and enhanced infiltration of CD8(+) T cells in the brain of Aß33-41NP-immunized APPPS1 mice, no clinical signs of severe autoimmune neuroinflammation were observed. CONCLUSIONS: Altogether, these results suggest that Aß-specific CD8(+) T cells are not major contributors to meningoencephalitis in response to Aß vaccination.

Engineered regulatory T cells coexpressing MHC class II:peptide complexes are efficient inhibitors of autoimmune T cell function and prevent the development of autoimmune arthritis.

Regulatory T cells (Tregs) are critical homeostatic components in preventing the development of autoimmunity, and are a major focus for their therapeutic potential for autoimmune diseases. To enhance the efficacy of Tregs in adoptive therapy, we developed a strategy for generating engineered Tregs that have the capacity to target autoimmune T cells in an Ag-specific manner. Using a retroviral expression system encoding Foxp3 and HLA-DR1 covalently linked to the immunodominant peptide of the autoantigen type II collagen (DR1-CII), naive T cells were engineered to become Tregs that express DR1-CII complexes on their surface. When these cells were tested for their ability to prevent the development of collagen induced arthritis, both the engineered DR1-CII-Foxp3 and Foxp3 only Tregs significantly reduced the severity and incidence of disease. However, the mechanism by which these two populations of Tregs inhibited disease differed significantly. Disease inhibition by the DR1-CII-Foxp3 Tregs was accompanied by significantly lower numbers of autoimmune CII-specific T cells in vivo and lower levels of autoantibodies in comparison with engineered Tregs expressing Foxp3 alone. In addition, the numbers of IFN-γ- and IL-17-expressing T cells in mice treated with DR1-CII-Foxp3 Tregs were also significantly reduced in comparison with mice treated with Foxp3 engineered Tregs or vector control cells. These data indicate that the coexpression of class II autoantigen-peptide complexes on Tregs provides these cells with a distinct capacity to regulate autoimmune T cell responses that differs from that used by conventional Tregs.

Adeno-associated virus-mediated gene transfer leads to persistent hepatitis B virus replication in mice expressing HLA-A2 and HLA-DR1 molecules.

Hepatitis B virus (HBV) persistence may be due to impaired HBV-specific immune responses being unable to eliminate efficiently or cure infected hepatocytes. The immune mechanisms that lead to HBV persistence have not been completely identified, and no appropriate animal model is available for such studies. Therefore, we established a chronic HBV infection model in a mouse strain with human leukocyte antigen A2/DR1 (HLA-A2/DR1) transgenes and an H-2 class I/class II knockout. The liver of these mice was transduced with adeno-associated virus serotype 2/8 (AAV2/8) carrying a replication-competent HBV DNA genome. In all AAV2/8-transduced mice, hepatitis B virus surface antigen, hepatitis B virus e antigen, and HBV DNA persisted in serum for at least 1 year. Viral replication intermediates and transcripts were detected in the livers of the AAV-injected mice. The hepatitis B core antigen was expressed in 60% of hepatocytes. No significant inflammation was observed in the liver. This was linked to a higher number of regulatory T cells in liver than in controls and a defect in HBV-specific functional T-cell responses. Despite the substantial tolerance resulting from expression of HBV antigens in hepatocytes, we succeeded in priming functional HBV-specific T-cell responses in peripheral tissues, which subsequently reached the liver. This AAV2/8-HBV-transduced HLA-A2/DR1 murine model recapitulates virological and immunological characteristics of chronic HBV infection, and it could be useful for the development of new treatments and immune-based therapies or therapeutic vaccines for chronic HBV infections.

Rheumatic heart disease in Uganda: the association between MHC class II HLA DR alleles and disease: a case control study.

BACKGROUND: Rheumatic heart disease (RHD), the only long term consequence of acute rheumatic fever, remains a leading cause of morbidity and mortality among young adults in Uganda. An inherited susceptibility to acute rheumatic fever centers around the major histocompatibility class II human leucocyte antigens. However, there is paucity of data from sub-Saharan Africa. This study compares the frequency of HLA class II DR alleles between RHD cases and normal controls in Uganda. METHODS: One hundred ninety-nine participants including 96 established RHD cases aged 5-60 years and 103 age and sex matched normal controls were recruited for participation. DNA was manually extracted from buffy coat samples and HLA analysis was performed. HLA-DR allelic frequency comparison between cases and controls were estimated using conditional logistic regression with 95% confidence intervals. P -values were corrected for multiple hypothesis testing. RESULTS: 199 participants (103 female, 51.8%) completed the study. The mean (SD) age in years for cases and controls were 29.6 (10.2) and 29(18), respectively. After conditional logistic regression and multiple hypothesis testing, HLA-DR1was associated with a decreased risk of RHD (OR = 0.42, CI 0.21-085, P = 0.01, Corrected P value (PC) = 0.09,) while HLA-DR11 was associated with increased risk of RHD (OR = 3.31, CI 1.57-6.97, P = <0.001, Pc < 0.001). No other significant associations were found. CONCLUSION: In this first study of HLA genetic susceptibility to RHD in Uganda, HLA- DR1 was more common in normal controls while HLA- DR11 was more common among RHD cases suggesting a disease susceptibility association. In future studies, high resolution HLA analysis and genome wide studies should be carried out to confirm this pattern.

Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4-HLA-DR1 complex.

Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K(D) = ∼200 µM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K(D) = 8.8 µM, compared with >400 µM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and ß2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.

The androgen receptor: a biologically relevant vaccine target for the treatment of prostate cancer.

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. However, while it has long been the primary molecular target of metastatic prostate cancer therapies, it has not been explored as an immunotherapeutic target. In particular, the AR ligand-binding domain (LBD) is a potentially attractive target, as it has an identical sequence among humans as well as among multiple species, providing a logical candidate for preclinical evaluation. In this report, we evaluated the immune and anti-tumor efficacy of a DNA vaccine targeting the AR LBD (pTVG-AR) in relevant rodent preclinical models. We found immunization of HHDII-DR1 mice, which express human HLA-A2 and HLA-DR1, with pTVG-AR augmented AR LBD HLA-A2-restricted peptide-specific, cytotoxic immune responses in vivo that could lyse human prostate cancer cells. Using an HLA-A2-expressing autochthonous model of prostate cancer, immunization with pTVG-AR augmented HLA-A2-restricted immune responses that could lyse syngeneic prostate tumor cells and led to a decrease in tumor burden and an increase in overall survival of tumor-bearing animals. Finally, immunization decreased prostate tumor growth in Copenhagen rats that was associated with a Th1-type immune response. These data show that the AR is as a prostate cancer immunological target antigen and that a DNA vaccine targeting the AR LBD is an attractive candidate for clinical evaluation.

High-throughput engineering and analysis of peptide binding to class II MHC.

Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.

Bidirectional binding of invariant chain peptides to an MHC class II molecule.

T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

Ontak reduces the immunosuppressive tumor environment and enhances successful therapeutic vaccination in HER-2/neu-tolerant mice.

Disrupting tumor-mediated mechanisms suppressing host immunity represents a novel approach to tumor immunotherapy. Depletion of regulatory T cells (Tregs) increases endogenous anti-tumor immunity and the efficacy of active immunotherapy in experimental tumor models. HLA-A2.1/HLA-DR1 (A2.1/DR1) × BALB- neuT+ (neuT+) triple transgenic mice represent an improvement over neuT+ mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. We questioned whether depletion of Tregs with Denileukin diftitox (Ontak) enhances the efficacy of a therapeutic vaccine consisting of HER-2(85-94) (p85) CTL and HER-2(776-790) (p776) Th peptides against the growth of TUBO.A2 transplantable tumor in male A2.1/DR1 × neuT+ Tg mice. While the therapeutic vaccine primed the tumor-reactive CD8+ CTLs and CD4+ effector T lymphocytes (Teffs) compartment, inducing activation, tumor infiltration, and tumor rejection or delay in tumor growth, treatment with Ontak 1 day prior to vaccination resulted in enhanced CD4+ and CD8+ T-cell-mediated vaccine-specific immune responses in the periphery. This was closely associated with greater infiltration and a striking change in the intratumor balance of Tregs and vaccine-specific CTLs/Teffs that directly correlated with markedly enhanced antitumor activity. The data suggest that Tregs control both CD4+ and CD8+ T-cell activity within the tumor, emphasize the importance of the intratumor ratio of vaccine-specific lymphocytes to Tregs, and demonstrate significant inversion of this ratio and correlation with tumor rejection during Ontak/vaccine immunotherapy.

Cutting edge: HLA-DM-mediated peptide exchange functions normally on MHC class II-peptide complexes that have been weakened by elimination of a conserved hydrogen bond.

The mechanism by which HLA-DM (DM) promotes exchange of peptides bound to HLA-DR (DR) is still unclear. We have shown that peptide interaction with DR1 can be considered a folding process as evidenced by cooperativity. However, in DM-mediated ligand exchange, prebound peptide release is noncooperative, which could be a function of the breaking of a critical interaction. The hydrogen bond (H-bond) between beta-chain His(81) and the peptide backbone at the -1 position is a candidate for such a target. In this study, we analyze the exchange of peptides bound to a DR1 mutant in which formation of this H-bond is impaired. We observe that DM still functions normally. However, as expected of a cooperative model, this H-bond contributes to the overall energetics of the complex and its disruption impacts the ability of the exchange ligand to fold with the binding groove into a stable complex.

An autoantigen-specific, highly restricted T cell repertoire infiltrates the arthritic joints of mice in an HLA-DR1 humanized mouse model of autoimmune arthritis.

Although it is clear that CD4(+) T cells play a major role in mediating the pathogenesis of autoimmunity, they often represent only a minor population at the site of inflammation in autoimmune diseases. To investigate the migration and specificity of autoimmune T cells to the inflammatory site, we used the collagen-induced arthritis model to determine the frequency, clonotype, and specificity of T cells that infiltrate arthritic joints. We demonstrate that despite the fact that CD4(+) T cells are a minor population of the synovial infiltrate, the CD4(+) T cells present are a highly selective subset of the TCR repertoire and, based on CDR3 length polymorphisms, have a limited clonality. Although a similar repertoire of type II collagen (CII)-specific TCR-BV8 and BV14-expressing T cells was found in peripheral lymphoid organs, the clonality of the TCR-BV8 and BV14 T cells that migrate to the arthritic joint generally made up a single CDR3 length. T cell hybridomas produced from these joint-derived cells revealed that many of these infiltrating T cells are CII specific, and the majority recognize mouse CII. These data suggest that despite being a minor population at the site of inflammation, autoantigen-specific T cells are selectively recruited and/or retained in the arthritic joint and may be playing a significant role in the pathogenesis of the autoimmune arthritis. In addition, this model may be very useful for studying the function in situ and the mechanism by which autoimmune T cells are recruited to the site of inflammation.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma.

BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

CD8+ T cells specific for the androgen receptor are common in patients with prostate cancer and are able to lyse prostate tumor cells.

The androgen receptor (AR) is a hormone receptor that plays a critical role in prostate cancer, and depletion of its ligand has long been the cornerstone of treatment for metastatic disease. Here, we evaluate the AR ligand-binding domain (LBD) as an immunological target, seeking to identify HLA-A2-restricted epitopes recognized by T cells in prostate cancer patients. Ten AR LBD-derived, HLA-A2-binding peptides were identified and ranked with respect to HLA-A2 affinity and were used to culture peptide-specific T cells from HLA-A2+ prostate cancer patients. These T-cell cultures identified peptide-specific T cells specific for all ten peptides in at least one patient, and T cells specific for peptides AR805 and AR811 were detected in over half of patients. Peptide-specific CD8+ T-cell clones were then isolated and characterized for prostate cancer cytotoxicity and cytokine expression, identifying that AR805 and AR811 CD8+ T-cell clones could lyse prostate cancer cells in an HLA-A2-restricted fashion, but only AR811 CTL had polyfunctional cytokine expression. Epitopes were confirmed using immunization studies in HLA-A2 transgenic mice, in which the AR LBD is an autologous antigen with an identical protein sequence, which showed that mice immunized with AR811 developed peptide-specific CTL that lyse HLA-A2+ prostate cancer cells. These data show that AR805 and AR811 are HLA-A2-restricted epitopes for which CTL can be commonly detected in prostate cancer patients. Moreover, CTL responses specific for AR811 can be elicited by direct immunization of A2/DR1 mice. These findings suggest that it may be possible to elicit an anti-prostate tumor immune response by augmenting CTL populations using AR LBD-based vaccines.

Weak or absent evidence for the association of HLA-DR antigens with risk of thyroid carcinoma: a meta-analysis of observational studies.

Inconsistent reports of associations between human leukocyte antigen (HLA)-DR and thyroid cancers exist. We conducted a comprehensive search of the PubMed, Scopus and Web of Science databases. Using random-effects modeling, subgroup analyses, meta-regression and prediction interval (PI) estimation, we combined the existing evidence from 13 studies (977 cases of thyroid cancer and 3735 controls). Only HLA-DR1 and HLA-DR11 were significantly associated; however, the evidence for HLA-DR11 came from only three studies while that for HLA-DR1 had large between-study heterogeneity. All the PIs estimated in the study straddled unity. Therefore, current evidence for the studied association is incomplete as well as uncertain. Attempts to include HLA-DR typing as a prognostic or therapeutic marker may be premature at this time.

Cutting edge: HLA-DM functions through a mechanism that does not require specific conserved hydrogen bonds in class II MHC-peptide complexes.

HLA-DM catalyzes peptide dissociation and exchange in class II MHC molecules through a mechanism that has been proposed to involve the disruption of specific components of the conserved hydrogen bond network in MHC-peptide complexes. HLA-DR1 molecules with alanine substitutions at each of the six conserved H- bonding positions were expressed in cells, and susceptibility to DM catalytic activity was evaluated by measuring the release of CLIP. The mutants alphaN62A, alphaN69A, alphaR76A, and betaH81A DR1 were fully susceptible to DM-mediated CLIP release, and betaN82A resulted in spontaneous release of CLIP. Using recombinant soluble DR1 molecules, the amino acid betaN82 was observed to contribute disproportionately in stabilizing peptide complexes. Remarkably, the catalytic potency of DM with each beta-chain mutant was equal to or greater than that observed with wild-type DR1. Our results support the conclusion that no individual component of the conserved hydrogen bond network plays an essential role in the DM catalytic mechanism.