IFN-gamma treatment increases insulin binding and MHC class I expression in erythroleukemia cells

We have investigated if interferon-gamma (IFN-gamma) treatment of human K562 tumor cells, which upregulates the expression of MHC class I antigens (MHC-I), simultaneously would influence insulin binding. Treatment of K562 cells with recombinant human IFN-gamma for 48 h caused a significant increase of insulin binding at 37 degrees C. Recombinant human tumor necrosis factor-alpha (TNF-alpha) alone had no effect but acted synergistically with IFN-gamma, leading to a two-fold increase of insulin binding. No change in affinity, number of binding sites or cell surface expression of insulin receptors (IR) after IFN-gamma treatment could be detected. The increased insulin binding observed at 37 degrees C was not seen at 4 degrees C, suggesting alteration of insulin internalization. The dose-response curve, as well as the time curve, for the increase in insulin binding after IFN-gamma treatment correlated with enhanced cell surface expression of MHC-I antigens. However, the correlation was not absolute. Our results show that IFN-gamma treatment alone or together with TNF-alpha, can alter the insulin binding to K562 cells without changing the expression or affinity of the IR. This correlates with the effect of IFN-gamma on MHC-I expression. These results support the findings that MHC-I molecules associate and interact with the IR at the cell surface.     

Regulation of MHC class I membrane expression by beta 2-microglobulin

MHC-I binding peptides and beta 2 microglobulin (beta 2-m) can upregulate the MHC-I heavy chain expression on certain peptide transporter mutant cells. We have further studied this with normal cells and non-mutant cell lines. No MHC-I upregulation was seen with normal, resting or activated T cells. On mouse cell lines P815 and B16, both peptides and human beta 2-m gave an additive upregulation response. With the human small cell lung carcinoma H82, an optimal HLA.A2 binding peptide (GILGFVFTL) gave an upregulation response, whereas beta 2-m alone or in combination with this peptide had no effect. However, beta 2-m potentiated the response of H82 cells to a slightly longer peptide. Using mutant RMA-S cells, it was found that both Brefeldin A (BFA) and chloroquine, but not leupeptin, inhibited MHC-I upregulation response to both peptide and beta 2-m. In contrast to chloroquine, BFA also gave a reduction of background membrane MHC-I expression, presumably due to a block in Golgi transport. Human beta 2-m, which binds to RMA-S cells, and which is known to internalize into endosomes, did not reappear on the cell surface. When Db on RMA-S cells was upregulated by human beta 2-m, the sensitivity of these cells to Db restricted CTL cells increased. Even if beta 2-m did not upregulate the overall MHC-I expression on normal cells, it may still quantitatively increase the expression of optimally presented peptides and endosomal recycling many be important in this process.     

NK cells can recognize different forms of class I MHC

NK recognition and lysis of targets are mediated by activation receptor(s) whose effects may be over-ridden by inhibitory receptors recognizing class I MHC on the target. Incubation of normal lymphoblasts with a peptide that can bind to their class I MHC renders them sensitive to lysis by syngeneic NK cells. By binding to class I MHC, the peptide alters or masks the target structure recognized by an inhibitory NK receptor(s). This target structure is most likely an "empty" dimer of class I heavy chain and beta2m as opposed to a "full" class I trimer formed by binding of specific peptide that is recognized by CTL.     

Orientation of the Ig domains of CD8 alpha beta relative to MHC class I

The cell surface glycoprotein CD8 functions as a coreceptor with the TCR for interaction with MHC class I. The cocrystal structure of the CD8 alpha alpha-MHC complex showed that one CD8 Ig domain provided the majority of the contact with MHC class I and that residue R4 of that domain contacted the alpha2 domain of MHC class I. We previously showed by mutational analysis that this residue was critical for binding to MHC class I. To determine which of the Ig domains for the CD8 alpha beta heterodimer would make the most contact with class I MHC, we expressed single-chain or dimeric forms of CD8 on COS-7 cells and measured the adhesion of MHC class I positive cells. We found that when one of the R4 residues was mutated in a CD8 alpha alpha homodimer binding comparable to that of wild type was observed, whereas a double R4 mutant severely impaired binding. However, when mutant CD8 alpha (R4K) was coexpressed with wild-type CD8 beta, binding was not observed. These results support the model in which it is CD8 alpha, not CD8 beta, that is making the most of the contact with MHC class I, including the alpha 2 domain. In addition, they demonstrate that a single-chain form of CD8 alpha alpha can bind to MHC class I.     

NK cells from human MHC class I (HLA-B) transgenic mice do not mediate hybrid resistance killing against parental nontransgenic cells

We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.     

Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

In this study the effects of two chitosan salts, namely chitosan hydrochloride and chitosan glutamate (0.5 and 1.5% w/v), on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cell monolayers, using the radioactive marker [14C]-mannitol, were investigated in a slightly acidic (pH 6.2) and neutral (pH 7.4) environment. Both salts are soluble in acidic conditions up to a concentration of 1.5% w/v and solutions of this strength, at a pH of 6.2, caused a pronounced lowering in the TEER of Caco-2 cell monolayers in the order of 70+/-1% (chitosan glutamate) and 77+/-3% (chitosan hydrochloride), 20 min after incubation started. In agreement with the TEER results the transport of the radioactive marker, [14C]-mannitol, was increased 25-fold (chitosan glutamate) and 36-fold (chitosan hydrochloride), respectively, at this pH. However, at a pH of 7.4 both salts are insoluble and prove to be ineffective since no reduction in the TEER values or increase in the transport of [14C]-mannitol were found. The results show that these chitosan salts are potent absorption enhancers in acidic environments. We conclude that there is a need for chitosan derivatives with increased solubility, especially at neutral and basic pH values, for use as absorption enhancers aimed at the delivery of therapeutic compounds in the more basic environment of the large intestine and colon.     

Major histocompatibility complex (MHC) class I recognition by natural killer cells

NK cells express clonally distributed receptors specific for MHC class I molecules. Structurally, these receptors belong to the C-type lectin superfamily in mouse and to the immunoglobulin superfamily in human. Functionally, they can be distinguished as inhibitory or stimulatory. Inhibitory receptors block NK cell-mediated cytotoxicity upon binding to HLA class I ligands. This function is mediated by phosphorylation of cytoplasmic tyrosines, which recruit the protein tyrosine phosphatase SHP-1. Stimulatory receptors also bind HLA class I, lack cytoplasmic tyrosine-based motifs, and trigger NK cell-mediated cytotoxicity. All these receptors are characterized by a limited diversity allowing for sensitive detection of loss of MHC class I molecules on autologous transformed and virally infected cells.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

Activation of human CD8+ alpha beta TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway

Human immune responses to M. tuberculosis are characterized by activation of multiple T cell subsets including CD4+, CD8+, and gammadelta T cells, and the role of CD8+ alphabeta TCR+ T cells in this response is poorly understood. Stimulation of T cells from healthy tuberculin skin test-positive persons with live M. tuberculosis-H37Ra or soluble M. tuberculosis Ags readily up-regulated IL-2Ralpha (CD25) expression on CD8+ T cells. Purified resting and activated CD8+ T cells produced IFN-gamma and proliferated to both M. tuberculosis bacilli and soluble mycobacterial Ags with monocytes as APC. Precursor frequency of mycobacterial Ag-specific CD8+ T cells by IFN-gamma enzyme-linked immunospot was 5-10-fold lower than the precursor frequency of CD4+ T cells, and IFN-gamma secretion by CD8+ T cells was 50-100-fold lower. CD8+ T cells secreted approximately 10-fold less IFN-gamma per cell than CD4+ T cells in response to mycobacterial Ags. CD8+ T cell responses to M. tuberculosis bacilli were blocked by anti-MHC class I antibody and required Ag processing. Processing of M. tuberculosis bacilli by monocytes for presentation to MHC class I-restricted CD8+ T cells was insensitive to brefeldin A treatment, which blocks the conventional MHC class I Ag-processing pathway. These results represent the first demonstration that human cells can process pathogen Ags via an alternate Ag-processing pathway for MHC class I and suggest a mechanism for participation of IFN-gamma-secreting CD8+ T cells in the human immune responses to M. tuberculosis.     

Differential presentation of the same MHC class I epitopes by fibroblasts and dendritic cells

Ag is presented to CTL as peptide associated with MHC class I molecules, which are present on most types of cells. We have investigated the presentation of Db-restricted lymphocytic choriomeningitis virus (LCMV) peptides by a fibroblast line (MC57) and a dendritic cell line (JawsII) to splenocytes from LCMV-immune C57BL/6 mice. We found that when LCMV-infected MC57 were used to restimulate the spleen cells, the resulting CTL line lost its ability to respond to the two dominant epitopes of the immune response to LCMV glycoprotein (gp)33 and nucleoprotein (np)396 but remained strongly lytic for targets coated with the subdominant gp276 epitope. In contrast, when LCMV-infected JawsII cells were used to restimulate the splenocytes, the resulting line continued to target gp33 and np396 but lost reactivity to gp276. When uninfected JawsII or MC57 cells were coated with peptides and used as stimulators, the resulting CTL lines continued to recognize all three epitopes, indicating that costimulatory or other potential innate differences in Ag presentation between the two cell lines are unlikely to account for the selective expansion of CTL specificities. When infected, both cell types produce similar levels of infectious LCMV, have similar levels of the NP and GP proteins from which np396 and gp33 are derived, and can be recognized by CTL specific for each of the three epitopes. These data indicate that in the generation of peptides for MHC-I binding and presentation to CTL, MC57 and JawsII process the same set of virus proteins in quantitatively different ways.     

Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells

A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.     

Self-peptides bound to the type I diabetes associated class II MHC molecules HLA-DQ1 and HLA-DQ8

Genetic susceptibility to several autoimmune disorders is associated with the expression of certain MHC class II alleles. Insight into the etiology of such diseases awaits the identification of the class II restriction elements and the possible pathogenic peptides. Towards these aims, self-peptides bound to HLA-DQ1 and HLA-DQ8, allotypes considered to be neutral and permissive respectively towards the development of insulin-dependent diabetes mellitus, are reported. These naturally processed peptides were isolated from immunoaffinity purified HLA-DQ molecules expressed in cultured B lymphocytes. The chromatographic profiles of the peptide repertoires are unique, whereas the size distributions exhibit general similarity to those reported for naturally processed self-peptides bound to HLA-DR. Twenty-eight individual peptides representing 10 nested sets were identified by combined Edman microsequencing and mass spectrometry. Peptide length varied from 13 to 74 amino acids. Source proteins included MHC molecules and other integral membrane proteins, as well as secretory, cytosolic and mitochondrial proteins. Promiscuous invariant chain peptides were identified among the self-peptides bound to HLA-DQ8. No dominant amino acid markers suggestive of particular enzymatic processing events were detected. Some structural features of DQ1 and DQ8 that may relate to the bound peptides are discussed. Peptide specificity was confirmed in binding assays with purified HLA-DQ and HLA-DR protein.     

Effect of propranolol and IFN-beta on the induction of MHC class II expression and cytokine production by IFN-gamma IN THP-1 human monocytic cells

Technologies for promoting quality of organizational services to the developmentally disabled have been evolving over the past several decades. Feedback reinforcement and, often, goal-setting, powerful change tools, generally are incorporated within behavioral interventions. Despite their promise, wide-scale application of these strategies often is impeded by natural and informal organizational contingencies. In an attempt to combat such impediments, a structure of interlocking contingencies was designed to train and support managers' provision of effective feedback to their subordinates, peers, and superiors. The system included formal scheduling of feedback, reinforcement and goal setting in a way that attempted to (a) minimize financial costs, time and effort; (b) empower participants by involving them in designing the specifics of the system; and (c) promote momentum by encouraging dense schedules of feedback. Within a period of less than 6 months, supervisors, managers, and professional specialists conducted brief audits and delivered nearly 9,000 written feedback reports to workers serving 129 clients, with the result that staff-client interactions and client engagement levels increased substantially. Future research should replicate these methods under more rigorous experimental conditions and formally assess some of the system's spillover into realms such as gains in clients' skill levels, and staff and public acceptance.     

Maturation of Qa-1b class I molecules requires beta 2-microglobulin but is TAP independent

Two conformationally distinct and stable forms of Qa-1b, one strongly associated with beta 2-microglobulin (beta 2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable beta 2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without beta 2m, there was no maturation of either form in cells from beta 2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires beta 2m for an initial folding step. However, beta 2m is not essential for subsequent processing of Qa-1b molecules.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Comparison of the roles of CD8 alpha alpha and CD8 alpha beta in interaction with MHC class I

The CD8 molecule is expressed either as an alpha/alpha homodimer or an alpha/beta heterodimer on thymocytes and cytotoxic T cells, and functions as a coreceptor in concert with TCR for binding the MHC class I/peptide complex. Although CD8alpha/beta heterodimers have been shown to be more effective coreceptors, the precise role of the beta-chain in TCR-mediated thymic maturation and T cell activation is not understood. To understand the role of CD8beta in mediating CD8/MHC class I interaction, we examined whether cell surface CD8alpha/beta heterodimer promotes better cell-cell adhesion with MHC class I than the CD8alpha/alpha homodimer. The abilities of different forms of CD8 to adhere to MHC class I were measured with a cell-cell binding assay. Using a wild-type CD8beta and -alpha, we found that CD8alphabeta heterodimers did not mediate greater cell-cell adhesion than CD8alphaalpha homodimers. Furthermore, we found that chimeric CD8beta-alpha homodimers afforded no detectable binding. These results do not support the idea that CD8alphabeta binding to MHC class I is greater than that of CD8alphaalpha. Rather, they point to an alternative explanation in which CD8beta may play an role in promoting CD8/TCR interaction and/or in signaling/regulatory pathways.