Intracellular HSP72 detection in HL60 cells using a flow cytometry system based on microfluidic analysis.

HSP72 is an important marker for various environmental stresses and diseases, and many researchers need to detect HSP72 levels in various cells. We have therefore developed an assay to monitor intracellular heat-shock protein 72 expression on a microfluidic Lab-on-a-chip platform. We established this method to detect HSP72 intracellularly by antibody staining with DNA counterstaining. The Lab-on-a-chip technology is simple and efficient when performing flow cytometric assays. By permeabilizing the cells for the delivery of antibodies, we were able to show HSP72 expression after 30 min heat-shock at 44 degrees C and then at various post-incubation times at 37 degrees C. We compared our method to a conventional flow cytometer and an enzyme immunoassay technique.

Clonal anergy induced in a CD8+ hapten-specific cytotoxic T-cell clone by an altered hapten-peptide ligand.

Clonal T-cell anergy has been proposed as a mechanism to ensure peripheral tolerance in vivo. Anergy has been reported to result from T cell activation with inappropriate antigen-presenting cells (APC) or, in the case of CD4+ T cells, also by altered peptide ligands. This study reveals that altered hapten ligands can also induce anergy in CD8+ T cells. The Kb-restricted, trinitrophenyl (TNP) specific cytotoxic T lymphocyte (CTL) clone E6 was found to lyse target cells presenting the TNP-modified peptides M4L-TNP (derived from mouse serum albumin) or O4TNP (derived from chicken ovalbumin), but not the corresponding dinitrophenol (DNP)-modified peptides. However, whereas M4L-DNP was found totally unreactive, O4DNP antagonistically inhibited M4L-TNP-mediated kill if expressed on the same target cell. Moreover, when presented alone on APC, O4DNP, but not M4L-DNP, induced anergy in clone E6 by preventing its subsequent proliferative response to M4L-TNP. The anergic state did not affect agonist-specific cytolysis or T-cell receptor (TCR) down-modulation by the anergized CTL, and proliferative responses were regained upon addition of interleukin (IL)-2 or IL-12 plus IL-18. These findings substantiate the similarity between hapten-and peptide-recognition by T cells. The induction as well as the reversal of anergy in CD8+ CTL may thus be of relevance not only in autoimmunity or tumour rejection, but also in contact hypersensitivity reactions to haptens.

Peptide immunization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity.

The identity of the effector T cell population involved in contact hypersensitivity is still questionable with evidence promoting both CD4+ or CD8+ T cells. Previous experimental studies have relied on the in vivo depletion of T cell subsets using antibody, or the use of knock-out mice with deficiencies in either CD4+ or CD8+ T cell-mediated immunity. To address the role of the class I- and class II-mediated pathways of T cell activation in contact hypersensitivity responses in mice with an intact immune system, we utilized various trinitrophenyl-derivatized peptides, which bind specifically with H-2Kb (major histocompatibility complex class I) or H-2I-Ab (major histocompatibility complex class II). The subcutaneous injection of major histocompatibility complex class II-specific, but not of class I-binding, hapten-derivatized peptides in incomplete Freund's adjuvant induced specific, albeit low, contact hypersensitivity responsiveness to trinitrochlorobenzene. When bone-marrow-derived dendritic cells, however, were pulsed with the same peptides and administered intradermally, the opposite result was observed, namely that the class I binding peptides induced contact hypersensitivity responses similar to that observed after epicutaneous trinitrochlorobenzene application. In contrast, dendritic cells pulsed with major histocompatibility complex class II binding peptides did not reproducibly sensitize for contact hypersensitivity responses. Surprisingly, both immunization protocols efficiently induced CD8+ effector T cells. These results support the notion that CD8+ T cells are the dominant effector population mediating contact hypersensitivity responsiveness and that the CD4+ T cell subset only contributes little if at all.

Regulation of transporter associated with antigen processing by phosphorylation.

The ATP-binding cassette transporter associated with antigen processing (TAP) is required for transport of antigenic peptides, generated by proteasome complexes in the cytoplasm, into the lumen of the endoplasmic reticulum where assembly with major histocompatibility complex class I molecules takes place. The TAP transporter is a heterodimer of TAP1 and TAP2. Here we show that both TAP1 and TAP2 are phosphorylated under physiological conditions. Phosphorylation induces formation of high molecular weight TAP complexes that contain TAP1, TAP2, tapasin, and class I heterodimers. In addition, a 43-kDa phosphoprotein, which appears to be a kinase, is contained in the phosphorylated TAP-containing complexes. Phosphorylated TAP complexes are able to bind peptides and ATP, however, they are not capable of transporting peptides. After de-phosphorylation, TAP complexes regain the ability to transport peptides. Interestingly, phosphorylation levels of TAP complexes induced by viral infection inversely correlates with a significant reduction in TAP-dependent peptide transport activity. Enhanced TAP phosphorylation appears to be one of several strategies that viruses have exploited to better escape from host immune surveillance. These results demonstrate that major histocompatibility complex class I antigen processing and presentation is modulated by reversible TAP phosphorylation, and implicate the importance of TAP phosphorylation in the regulation of cytotoxic immune response.

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d).

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.

Impaired immunoproteasome assembly and immune responses in PA28-/- mice.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Partial agonism and independent modulation of T cell receptor and CD8 in hapten-specific cytotoxic T cells.

We recently demonstrated antagonism for hapten-reactive T cells by altered hapten ligands. Here we investigated partial peptide- or hapten-agonism and effects of antigen stimulation on the expression of TCR and the CD8 coreceptor using a set of DNP- or TNP-peptide-induced, H-2Kb-restricted mouse CTL clones. Various Kb-binding TNP- and DNP-peptides acted as partial agonists, cross-reactively stimulating individual clones for cytotoxicity and IFN-gamma secretion, but failing to induce proliferation or TNF-alpha production. Full agonism, i.e. activation of all possible functions, was usually restricted to those hapten-peptide combinations used for the induction of the respective clones. Our data imply distinctive kinetic optima for TCR antigen contacts in the induction of the various T cell effector functions. Down-regulation of TCR was efficiently induced by full, but with one exception not by partial, agonists, indicating the independence of cytotoxicity or IFN-gamma secretion from TCR modulation. On the other hand, a reduction of TCR expression induced by full agonists was usually not accompanied by synchronous down-modulation of CD8 as reported by others for human T cells. In fact, three of four full agonists and all partial agonists markedly enhanced rather than reduced the expression of CD8. Increased CD8 surface levels enhanced cytolytic potential and increased cross-reactivity patterns of individual clones. Brefeldin A blocked this CD8 induction by partial agonists, and in the case of full agonists resulted in a parallel reduction of both, TCR and CD8. Thus, antigenic stimulation of mouse T cells initially down-modulates CD8 together with TCR, but the loss of coreceptor is over-compensated by a signal for increased CD8 export.

Altered hapten ligands antagonize trinitrophenyl-specific cytotoxic T cells and block internalization of hapten-specific receptors.

Low molecular chemicals (haptens) frequently cause T cell-mediated adverse immune reactions. Our previous work provided evidence that hapten-specific T cells, in analogy to those specific for nominal peptide antigens, direct their TCR towards hapten-modified, MHC-associated peptides. We now demonstrate that trinitrophenyl (TNP)-specific, class I MHC-restricted CTL from mice may exhibit exquisite specificity for subtle structural details of these hapten determinants, surpassing even the specificity of immunoglobulins. More importantly, these CTL could be antagonized by ligands altered either in their peptide sequence or in their hapten structure. The system was employed to examine the molecular basis of T cell antagonism. Whereas agonists resulted in a dose-dependent downregulation of TCR in different mouse T cell clones, antagonistic peptides totally failed to do so despite engaging the specific TCR. Moreover, simultaneous presentation of antagonist and agonist on the same antigen presenting cell prevented TCR internalization. No signs of anergy or functional receptor inactivation were observed in CTL treated with antagonist-loaded target cells. Based on a serial triggering model of T cell activation, our data favor a model in which antagonists block T cell functions by competitively engaging the specific TCR in unproductive interactions.