High-Density SNP Screening of the Major Histocompatibility Complex in Systemic Lupus Erythematosus Demonstrates Strong Evidence for Independent Susceptibility Regions

A substantial genetic contribution to systemic lupus erythematosus (SLE) risk is conferred by major histocompatibility complex (MHC) gene(s) on chromosome 6p21. Previous studies in SLE have lacked statistical power and genetic resolution to fully define MHC influences. We characterized 1,610 Caucasian SLE cases and 1,470 parents for 1,974 MHC SNPs, the highly polymorphic HLA-DRB1 locus, and a panel of ancestry informative markers. Single-marker analyses revealed strong signals for SNPs within several MHC regions, as well as with HLA-DRB1 (global p = 9.99×10⁻¹⁶). The most strongly associated DRB1 alleles were: *0301 (odds ratio, OR = 2.21, p = 2.53×10⁻¹²), *1401 (OR = 0.50, p = 0.0002), and *1501 (OR = 1.39, p = 0.0032). The MHC region SNP demonstrating the strongest evidence of association with SLE was rs3117103, with OR = 2.44 and p = 2.80×10⁻¹³. Conditional haplotype and stepwise logistic regression analyses identified strong evidence for association between SLE and the extended class I, class I, class III, class II, and the extended class II MHC regions. Sequential removal of SLE–associated DRB1 haplotypes revealed independent effects due to variation within OR2H2 (extended class I, rs362521, p = 0.006), CREBL1 (class III, rs8283, p = 0.01), and DQB2 (class II, rs7769979, p = 0.003, and rs10947345, p = 0.0004). Further, conditional haplotype analyses demonstrated that variation within MICB (class I, rs3828903, p = 0.006) also contributes to SLE risk independent of HLA-DRB1*0301. Our results for the first time delineate with high resolution several MHC regions with independent contributions to SLE risk. We provide a list of candidate variants based on biologic and functional considerations that may be causally related to SLE risk and warrant further investigation.     

The Effect of Lung Cancer on Cytokine Expression in Peripheral Blood Mononuclear Cells

The purpose of this study is to evaluate cytokine expression by peripheral blood mononuclear cells (PBMC) from stage I lung cancer patients and to confirm these expression patterns by exposing PBMCs to lung cancer cells in vitro. Five altered cytokines in stage I lung cancer patients (CCL3, IL8, IL1β, CXCL10, sIL2Rα) were identified in plasma from subjects (n = 15) before and after resection using a 30-plex panel protein assay. Gene expression studies using quantitative RT-qPCR were performed on PBMCs from stage I lung cancer patients (n = 62) before and after resection, and compared to non-cancer patients (n = 32) before and after surgery for benign disease. Co-culture experiments that exposed healthy donor PBMCs to lung cancer cells in vitro were performed to evaluate the effect on PBMC cytokine expression. PBMC gene expression of CCL3, IL8 and IL1β was higher in lung cancer patients compared to the same patients at each of four sequential timepoints after removal of their tumors, while CXCL10 and IL2Rα were essentially unchanged. This pattern was also detected when lung cancer patients were compared to non-cancer patients. When non-cancer patients underwent surgery for benign diseases, these cytokine expression changes were not demonstrable. Lung cancer cell lines, but not benign bronchial epithelial cells, induced similar changes in cytokine gene and protein expression by healthy donor PBMCs in an in vitro co-culture system. We conclude that PBMCs from stage I lung cancer patients possess distinct cytokine expression patterns compared to both non-cancer patients, and lung cancer patients following tumor removal. These expression patterns are replicated by healthy donor PBMCs exposed to lung cancer cell lines, but not benign bronchial epithelial cells in vitro. These findings have implications for understanding the immune response to lung cancer.     

Refinement of the MHC Risk Map in a Scandinavian Primary Sclerosing Cholangitis Population

Background

Genetic variants within the major histocompatibility complex (MHC) represent the strongest genetic susceptibility factors for primary sclerosing cholangitis (PSC). Identifying the causal variants within this genetic complex represents a major challenge due to strong linkage disequilibrium and an overall high physical density of candidate variants. We aimed to refine the MHC association in a geographically restricted PSC patient panel.

Methodology/Principal Findings

A total of 365 PSC cases and 368 healthy controls of Scandinavian ancestry were included in the study. We incorporated data from HLA typing (HLA-A, -B, -C, -DRB3, -DRB1, -DQB1) and single nucleotide polymorphisms across the MHC (n = 18,644; genotyped and imputed) alongside previously suggested PSC risk determinants in the MHC, i.e. amino acid variation of DRβ, a MICA microsatellite polymorphism and HLA-C and HLA-B according to their ligand properties for killer immunoglobulin-like receptors. Breakdowns of the association signal by unconditional and conditional logistic regression analyses demarcated multiple PSC associated MHC haplotypes, and for eight of these classical HLA class I and II alleles represented the strongest association. A novel independent risk locus was detected near NOTCH4 in the HLA class III region, tagged by rs116212904 (odds ratio [95% confidence interval] = 2.32 [1.80, 3.00], P = 1.35×10⁻¹¹).

Conclusions/Significance

Our study shows that classical HLA class I and II alleles, predominantly at HLA-B and HLA-DRB1, are the main risk factors for PSC in the MHC. In addition, the present assessments demonstrated for the first time an association near NOTCH4 in the HLA class III region.     

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.     

LILRB2 Interaction with HLA Class I Correlates with Control of HIV-1 Infection

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8⁺ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10⁻²). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10⁻¹¹–10⁻⁹) and African (p = 10⁻⁵–10⁻³) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement.     

Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4⁺ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii⁻ cells may present peptides not presented by Ii⁺ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii⁺ cells, but will not be tolerant to novel peptides presented by Ii⁻ cells, we generated MHC II vaccines to activate cancer patients'' T cells. The vaccines are Ii⁻ tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii⁺ and Ii⁻ MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii⁻ cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii⁻ and Ii⁺ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii⁻ cells are not presented by Ii⁺ cells, and are derived from source proteins not used by Ii⁺ cells. Peptides from Ii⁻ cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7⁺ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II⁺ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC)¹ class I, molecules capable of activating CD8⁺ T-cells that directly kill tumor cells (1, 2). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4⁺ T-cells despite compelling evidence that Type 1 CD4⁺ T helper cells facilitate the optimal activation of CD8⁺ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease.Activation of CD4⁺ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4⁺ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3, 4). As a result, tumor-reactive CD4⁺ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated “MHC II cancer vaccines” that consist of Ii⁻ tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II⁺Ii⁻CD80⁺ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4⁺ T cells produce IFNγ and provide help to CD8⁺ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4⁺ Th1 cells that facilitate antitumor immunity. In vitro (5) and in vivo (5–7) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4⁺ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4⁺ T cells with a distinct repertoire of T cell receptors (8–12). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13, 14), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4⁺ T cells (14, 15).We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II⁺Ii⁻ and MHC II⁺Ii⁺ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7⁺ breast cancer cells are unique to Ii⁻ cells and are derived from source proteins not used by Ii⁺ cells. Ii⁻ peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II⁺Ii⁻ tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors     

Separate Developmental Programs for HLA-A and -B Cell Surface Expression during Differentiation from Embryonic Stem Cells to Lymphocytes,Adipocytes and Osteoblasts

A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017). Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4⁺ and CD8⁺ T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4⁺ and CD8⁺ T cells, respectively). Thus different HLA loci are differentially regulated during differentiation of stem cells.     

The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8⁺ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 γ₁-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV γ₂-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8⁺ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.     

Alloreactive T-cell clones. Ly phenotypes predict both function and specificity for major histocompatibility complex products

We have studied the association of Ly phenotype with function and specificity for major histocompatibility complex (MHC) products by examining the properties of 21 T-cell clones derived from B10 anti-B 10.D2 and B10.A anti-B10.D2 mixed lymphocyte cultures (MLC). T cells were selected after MLC solely on the basis of Ly phenotype, cloned by limiting dilution, and tested for stability of Ly phenotype, function and specificity for class I or class II MHC products. Sixteen Ly-1⁺2^– and five Ly-1^–2⁺ T-cell clones were tested. The clones selected for the Ly-1 ⁺2^– phenotype maintained this phenotype, expressed helper but not lytic function, and recognized class II MHC products (I-A^d or I-Ed). All Ly-1^–2^– clones maintained this phenotype, possessed cytolytic but not helper activity, and recognized class I MHC products (D^d and L^d). Our data therefore confirm at the clonal level the original observations of a remarkably consistent correlation between Ly markers, MHC specificity, and. function. They suggest that the expression of Ly antigens on T-cell clones forms part of a genetic program for each of these specialized cells that also determines their function and MHC specificity.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - TCGF T cell growth factor (Interleukin-2) - Con A Concanavalin A - DME Dulbecco's modified Eagle's medium - PHA phytohaemagglutinin - LPS lipopolysaccharide - TRF-C T cell replacing factor required for induction of cytolytic cells from thymocytes - PBS phosphate-buffered saline (pH 7.4)     

Treatment of ovarian cancer cell lines with 5-aza-2′-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules

Purpose  To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8⁺ T cells. Methods  Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC) for 3–7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-γ ELISpot analysis. Results  DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1–3 μM for CTA and up to 10 μM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8⁺ T cells. Conclusions  These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.     

Increased Circulating Th17 Cells after Transarterial Chemoembolization Correlate with Improved Survival in Stage III Hepatocellular Carcinoma: A Prospective Study

Transarterial chemoembolization (TACE) has therapeutic effects in patients with unresectable hepatocellular carcinoma (HCC), but its impact on the cellular immune response during disease progression is largely unknown. Here we conducted a prospective study to evaluate the effect of TACE on immune status and to identify prognostic immune markers governing treatment success. In this study, 51 stage III HCC patients, 28 stage I HCC patients (TNM classification) and 20 healthy donors were enrolled. Flow cytometry and cytometric bead array were used to evaluate the circulating immune cell subsets, including CD4⁺ T cells (Th1, Th17 and Treg cells), CD8⁺ T cells, NK cells, and NKT cells, and plasma cytokines before TACE and 30 days after TACE. Interestingly, among those immune parameters, the frequency of circulating Th17 cells was higher in stage III HCC patients than in stage I HCC patients (P = 0.015) and healthy donors (P<0.001). Moreover, an increased frequency of circulating Th17 cells was observed 30 days after TACE (Th17_D30) compared with the baseline level (P = 0.036). Kaplan-Meier analysis demonstrated that Th17_D30 was positively associated with overall survival (OS; P = 0.007) and time to progression (TTP; P = 0.009). Multivariate Cox analysis revealed that Th17_D30 was an independent prognostic factor for OS (HR = 0.317, P = 0.032) and TTP (HR = 0.304, P = 0.010). These results provide a potential prognostic marker for stage III HCC patients undergoing TACE and may be useful for identifying patients who can benefit from adjuvant immunotherapies.     

Distinct Expression Levels and Patterns of Stem Cell Marker,Aldehyde Dehydrogenase Isoform 1 (ALDH1), in Human Epithelial Cancers

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn''t significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDH^br tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.     

P2X7 Receptor Activation Impairs Exogenous MHC Class I Oligopeptides Presentation in Antigen Presenting Cells

Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8⁺ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8⁺ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8⁺ T cell immunity.     

Cortisol Patterns Are Associated with T Cell Activation in HIV

Objective

The level of T cell activation in untreated HIV disease is strongly and independently associated with risk of immunologic and clinical progression. The factors that influence the level of activation, however, are not fully defined. Since endogenous glucocorticoids are important in regulating inflammation, we sought to determine whether less optimal diurnal cortisol patterns are associated with greater T cell activation.

Methods

We studied 128 HIV-infected adults who were not on treatment and had a CD4⁺ T cell count above 250 cells/µl. We assessed T cell activation by CD38 expression using flow cytometry, and diurnal cortisol was assessed with salivary measurements.

Results

Lower waking cortisol levels correlated with greater T cell immune activation, measured by CD38 mean fluorescent intensity, on CD4⁺ T cells (r = −0.26, p = 0.006). Participants with lower waking cortisol also showed a trend toward greater activation on CD8⁺ T cells (r = −0.17, p = 0.08). A greater diurnal decline in cortisol, usually considered a healthy pattern, correlated with less CD4⁺ (r = 0.24, p = 0.018) and CD8⁺ (r = 0.24, p = 0.017) activation.

Conclusions

These data suggest that the hypothalamic-pituitary-adrenal (HPA) axis contributes to the regulation of T cell activation in HIV. This may represent an important pathway through which psychological states and the HPA axis influence progression of HIV.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Reduction of the CD16?CD56bright NK Cell Subset Precedes NK Cell Dysfunction in Prostate Cancer

Background

Natural cytotoxicity, mediated by natural killer (NK) cells plays an important role in the inhibition and elimination of malignant tumor cells. To investigate the immunoregulatory role of NK cells and their potential as diagnostic markers, NK cell activity (NKA) was analyzed in prostate cancer (PCa) patients with particular focus on NK cell subset distribution.

Methods

Prospective data of NKA and NK cell subset distribution patterns were measured from 51 patients initially diagnosed with PCa and 54 healthy controls. NKA was represented by IFN-γ levels after stimulation of the peripheral blood with Promoca®. To determine the distribution of NK cell subsets, PBMCs were stained with fluorochrome-conjugated monoclonal antibodies. Then, CD16⁺CD56^dim and CD16⁻CD56^bright cells gated on CD56⁺CD3⁻ cells were analyzed using a flow-cytometer.

Results

NKA and the proportion of CD56^bright cells were significantly lower in PCa patients compared to controls (430.9 pg/ml vs. 975.2 pg/ml and 2.3% vs. 3.8%, respectively; p<0.001). Both tended to gradually decrease according to cancer stage progression (p for trend = 0.001). A significantly higher CD56^dim-to-CD56^bright cell ratio was observed in PCa patients (41.8 vs. 30.3; p<0.001) along with a gradual increase according to cancer stage progression (p for trend = 0.001), implying a significant reduction of CD56^bright cells in relation to the alteration of CD56^dim cells. The sensitivity and the specificity of NKA regarding PCa detection were 72% and 74%, respectively (best cut-off value at 530.9 pg/ml, AUC = 0.786).

Conclusions

Reduction of CD56^bright cells may precede NK cell dysfunction, leading to impaired cytotoxicity against PCa cells. These observations may explain one of the mechanisms behind NK cell dysfunction observed in PCa microenvironment and lend support to the development of future cancer immunotherapeutic strategies.