Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Intrauterine growth restriction and absent or reverse end-diastolic blood flow in umbilical artery (Doppler class II or III): A retrospective study of short- and long-term fetal morbidity and mortality.

OBJECTIVE: Absent or reverse end-diastolic flow (Doppler II/III) in umbilical artery is correlated with poor perinatal outcome, particularly in intrauterine growth restricted (IUGR) fetuses. The optimal timing of delivery is still controversial. We studied the short- and long-term morbidity and mortality among these children associated with our defined management. STUDY DESIGN: Sixty-nine IUGR fetuses with umbilical Doppler II/III were divided into three groups; Group 1, severe early IUGR, no therapeutic intervention (n = 7); Group 2, fetuses with pathological biophysical profile, immediate delivery (n = 35); Group 3, fetuses for which expectant management had been decided (n = 27). RESULTS: In Group 1, stillbirth was observed after a mean delay of 6.3 days. Group 2 delivered at an average of 31.6 weeks and two died in the neonatal period (6%). In Group 3 after a mean delay of 8 days, average gestational age at delivery was 31.7 weeks; two intra uterine and four perinatal deaths were observed (22%). Long-term follow-up revealed no sequelae in 25/31 (81%) and 15/18 (83%), and major handicap occurred in 1 (3%) and 2 patients (11%), respectively, for Groups 2 and 3. CONCLUSION: Fetal mortality was observed in 22% of this high risk group. After a mean period of follow-up of 5 years, 82% of infants showed no sequelae. According to our management, IUGR associated with umbilical Doppler II or III does not show any benefit from an expectant management in term of long-term morbidity.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

Fine mapping of human cytotoxic T lymphocyte (CTL) responses against hepatitis C virus (HCV) is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC) class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS)/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS) 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂). In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Bacterial and viral superantigens: roles in autoimmunity?

Superantigens are bacterial, viral, or retroviral proteins which can activate specifically a large proportion of T cells. In contrast with classical peptide antigen recognition, superantigens do not require processing to small peptides but act as complete or partially processed proteins. They can bind to major histocompatibility complex class II molecules and stimulate T cells expressing particular T cell receptor V beta chains. The other polymorphic parts of the T cell receptor, which are crucial for classical antigen recognition, are not important for this interaction. When this strategy is used a large proportion of the host immune system can be activated shortly after infection. The activated cells have a wide variety of antigen specificities. The ability to stimulate polyclonal B (IgG) as well as T cell responses raises possibilities of a role for superantigens in the induction of autoimmune diseases. Superantigens have been a great tool in the hands of immunologists in unravelling some of the basic mechanisms of tolerance and immunity.

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition.

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

Association of the hemochromatosis gene with pazopanib-induced transaminase elevation in renal cell carcinoma.

BACKGROUND & AIMS: Pazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC.¦METHODS: Data from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels.¦RESULTS: Of 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure.¦CONCLUSIONS: The rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.

A Role for cis Interaction between the Inhibitory Ly49A receptor and MHC class I for natural killer cell education.

Natural killer (NK) cells show enhanced functional competence when they express inhibitory receptors specific for inherited major histocompatibility complex class I (MHC-I) molecules. Current models imply that NK cell education requires an interaction of inhibitory receptors with MHC-I expressed on other cells. However, the inhibitory Ly49A receptor can also bind MHC-I ligand on the NK cell itself (in cis). Here we describe a Ly49A variant, which can engage MHC-I expressed on other cells but not in cis. Even though this variant inhibited NK cell effector function, it failed to educate NK cells. The association with MHC-I in cis sequestered wild-type Ly49A, and this was found to relieve NK cells from a suppressive effect of unengaged Ly49A. These data explain how inhibitory MHC-I receptors can facilitate NK cell activation. They dissociate classical inhibitory from educating functions of Ly49A and suggest that cis interaction of Ly49A is necessary for NK cell education.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

Inflammation And Autoimmune Responses Are Independent Of Peripheral Mhc Class Ii Expression Driven By Ciita Piv In Collagen Induced Arthritis

Background In rheumatoid arthritis (RA), non-professional antigen presenting cells (APCs) such as fi broblast-like synoviocytes (FLS) can express MHC class II (MHCII) molecules and function as non-professional APCs in vitro.Objective To examine the regulation of MHCII expression in FLS and to investigate the role of FLS as non-professional APCs in collagen-induced arthritis (CIA). Methods Expression of MHCII, CIITA and Ciita isoforms pI, pIII and pIV was examined by RT-qPCR, immunohistochemistry and fl ow cytometry in human synovial tissues, arthritic mouse joints and human as well as mouse FLS. CIA was induced in mice knockout for the isoform IV of Ciita (pIV-/-), in pIV-/- mice transgenic for CIITA in the thymus (pIV-/- K14 CIITA) and in control littermates in the DBA/1 background by immunising with bovine collagen type II (CII) in complete Freund's adjuvant.Results HLA-DRA, total CIITA and CIITA pIII mRNA levels were signifi cantly increased in the synovial tissues from RA compared to osteoarthritis patients. Human FLS expressed surface MHCII via CIITA pIII and pIV, while MHCII expression in murine FLS was entirely mediated by pIV. pIV-/- mice lacked both inducible MHCII expression on non-professional APCs including FLS, and in the thymic cortex. The thymic defect in pIV-/- mice impaired CD4+ positive selection, thus protecting pIV-/- mice from CIA by preventing CD4+ T cells immune responses against CII and blocking the release of IFN-γ and IL-17 in ex vivo stimulated lymph node cells. The production of T dependent, arthritogenic anti-CII antibodies was also impaired in pIV-/- mice. A normal thymic expression of MHCII and CD4+ T cell repertoire was obtained in pIV-/- K14 CIITA Tg mice. Immune responses against CII were restored in pIV-/- K14 CIITA Tg mice, as well as the arthritis incidence and clinical severity despite the lack of MHCII expression by mouse FLS. At histology, infl ammation andneutrophils infi ltration scores were not reduced in pIV-/- K14 CIITA Tg mice, while the bone erosion score was signifi cantly lower than in controls.Conclusion Over expression of MHCII is tightly correlated with CIITA pIII in the arthritic human synovium. MHCII is induced via CIITA pIII and pIV in human FLS. In the mouse, MHCII expression in the thymic cortex and in FLS is strictly dependent upon Ciita pIV. The lack of Ciita pIV in the periphery of pIV-/- K14 CIITA Tg mice lowered the bone erosion score but did not signifi cantly protect from infl ammation and autoimmune responses in CIA.

Long maximal incremental tests accurately assess aerobic fitness in class II and III obese men.

This study aimed to compare two different maximal incremental tests with different time durations [a maximal incremental ramp test with a short time duration (8-12 min) (STest) and a maximal incremental test with a longer time duration (20-25 min) (LTest)] to investigate whether an LTest accurately assesses aerobic fitness in class II and III obese men. Twenty obese men (BMI≥35 kg.m-2) without secondary pathologies (mean±SE; 36.7±1.9 yr; 41.8±0.7 kg*m-2) completed an STest (warm-up: 40 W; increment: 20 W*min-1) and an LTest [warm-up: 20% of the peak power output (PPO) reached during the STest; increment: 10% PPO every 5 min until 70% PPO was reached or until the respiratory exchange ratio reached 1.0, followed by 15 W.min-1 until exhaustion] on a cycle-ergometer to assess the peak oxygen uptake [Formula: see text] and peak heart rate (HRpeak) of each test. There were no significant differences in [Formula: see text] (STest: 3.1±0.1 L*min-1; LTest: 3.0±0.1 L*min-1) and HRpeak (STest: 174±4 bpm; LTest: 173±4 bpm) between the two tests. Bland-Altman plot analyses showed good agreement and Pearson product-moment and intra-class correlation coefficients showed a strong correlation between [Formula: see text] (r=0.81 for both; p≤0.001) and HRpeak (r=0.95 for both; p≤0.001) during both tests. [Formula: see text] and HRpeak assessments were not compromised by test duration in class II and III obese men. Therefore, we suggest that the LTest is a feasible test that accurately assesses aerobic fitness and may allow for the exercise intensity prescription and individualization that will lead to improved therapeutic approaches in treating obesity and severe obesity.

Evaluation of Fly Ash Concrete Durability Containing Class II Durability Aggregates, July 1986

Fly ash was used in this evaluation study to replace 15% of the cement in Class C-3 concrete paving mixes. One Class "c" ash from Iowa approved sources was examined in each mix. Substitution rate was based on 1 to 1 basis, for each pound of cement removed 1.0 pound of ash was added. The freeze/thaw durability of the concrete studied was not adversely affected by the presence of fly ash. This study reveals that the durability of the concrete test specimens made with Class II durability aggregates was slightly increased in all cases by the substitution of cement with 15% Class "c" fly ash. In all cases durability factors either remained the same or slightly improved except for one case where the durability factor decreased from 36 to 34. The expansion decreased in all cases.

Evaluation of Fly Ash Concrete Durability Containing Class II Durability Aggregates, July 1986

Fly ash was used in this evaluation study to replace 15% of the cement in Class C-3 concrete paving mixes. One Class "c" ash from Iowa approved sources was examined in each mix. Substitution rate was based on 1 to 1 basis, for each pound of cement removed 1.0 pound of ash was added. The freeze/thaw durability of the concrete studied was not adversely affected by the presence of fly ash. This study reveals that the durability of the concrete test specimens made with Class II durability aggregates was slightly increased in all cases by the substitution of cement with 15% Class "c" fly ash. In all cases durability factors either remained the same or slightly improved except for one case where the durability factor decreased from 36 to 34. The expansion decreased in all cases.

Functional analysis of monocyte MHC class II compartments.

Circulating monocytes, as dendritic cell and macrophage precursors, exhibit several functions usually associated with antigen-presenting cells, such as phagocytosis and presence of endosomal/lysosomal degradative compartments particularly enriched in Lamp-1, MHC class II molecules, and other proteins related to antigen processing and MHC class II loading [MHC class II compartments (MIICs)]. Ultrastructural analysis of these organelles indicates that, differently from the multivesicular bodies present in dendritic cells, in monocytes the MIICs are characterized by a single perimetral membrane surrounding an electron-dense core. Analysis of their content reveals enrichment in myeloperoxidase, an enzyme classically associated with azurophilic granules in granulocytes and mast cell secretory lysosomes. Elevation in intracellular free calcium levels in monocytes induced secretion of beta-hexosaminidase, cathepsins, and myeloperoxidase in the extracellular milieu; surface up-regulation of MHC class II molecules; and appearance of lysosomal resident proteins. The Ca(2+)-regulated surface transport mechanism of MHC class II molecules observed in monocytes is different from the tubulovesicular organization of the multivesicular bodies previously reported in dendritic cells and macrophages. Hence, in monocytes, MHC class II-enriched organelles combine degradative functions typical of lysosomes and regulated secretion typical of secretory lysosomes. More important, Ca(2+)-mediated up-regulation of surface MHC class II molecules is accompanied by extracellular release of lysosomal resident enzymes.

Clinical consequences of sensitisation to minor histocompatibility antigens before allogeneic bone marrow transplantation.

To study sensitisation to minor histocompatibility antigens (mHag) before and after BMT, we measured antidonor CTL activity in five patients who had rejected their graft, and in a control group of 10 leukemic patients who engrafted without complications. All patients were transplanted with marrow from an HLA-identical sibling. Fourteen patients were conditioned with cyclophosphamide (120 mg/kg) and TBI (1350 cGy) and received a T cell-depleted graft, while one patient with aplastic anaemia received cyclophosphamide alone and unmanipulated marrow. Before transplantation, anti-donor CTL activity was detected in two of the 15 patients. These patients rejected their grafts at days 21 and 58, respectively. In the other three patients who rejected their grafts at days 41, 60 and 250, CTL activity was found only after transplantation. In contrast, no anti-donor CTLs could be detected at any time in the 10 patients who engrafted permanently. We have identified some of the mHags recognised during graft rejection by cloning and subsequent specificity analysis of the recipient CTLs. In the patient who rejected at day 41 without detectable immunisation before BMT, the response was directed against HA-1, a minor antigen known to play a role in GVHD. In the other combinations, a significant part of the CTL activity was directed against the male antigen H-Y. In the patient who rejected the marrow of her HLA-identical brother at day 250, two clones recognised H-Y, while five others recognised at least three distinct autosomal mHags. This patient had an HLA-identical sister who expressed only one autosomal mHag that had been recognised by one single T cell clone. After re-transplantation with the marrow of this second donor, the CTL activity could no longer be detected and the patient engrafted without further complications.

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.