T cell recognition of major histocompatibility complex class II complexes with invariant chain processing intermediates.

Peptides from the lumenal portion of invariant chain (Ii) spanning residues 80-106 (class II-associated Ii peptide [CLIP]) are found in association with several mouse and human major histocompatibility complex (MHC) class II allelic variants in wild-type and presentation-deficient mutant cells. The ready detection of these complexes suggests that such an intermediate is essential to the MHC class II processing pathway. In this study, we demonstrate that T cells recognize CLIP/MHC class II complexes on the surface of normal and mutant cells in a manner indistinguishable from that of nominal antigenic peptides. Surprisingly, T cell hybrids specific for human CLIP bound to murine MHC class II molecule I-Ab and a new monoclonal antibody 30-2 with the same specificity, recognize two independent epitopes expressed on this peptide/class II complex. T cell recognition is dependent on a Gln residue (position 100) in CLIP, whereas the 30-2 antibody recognizes a Lys residue-at position 90. These two residues flank the 91-99 sequence that is conserved among human, mouse, and rat Ii, potentially representing an MHC class II-binding site. Our results suggest that the COOH-terminal portion of CLIP that includes TCR contact residue Gln 100 binds in the groove of I-Ab molecule. Moreover, both T cells and the antibody recognize I-Ab complexed with larger Ii processing intermediates such as the approximately 12-kD small leupeptin-induced protein (SLIP) fragments. Thus, SLIP fragments contain a CLIP region bound to MHC class II molecule in a conformation identical to that of a free CLIP peptide. Finally, our data suggest that SLIP/MHC class II complexes are precursors of CLIP/MHC class II complexes.

Major histocompatibility complex class II compartments in human and mouse B lymphoblasts represent conventional endocytic compartments.

In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II-peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II- peptide complexes, as shown by using an antibody against an abundant endogenous class II-peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.

Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins.

We have identified and cloned a novel gene, trp-p8, by screening a prostate-specific subtracted cDNA library. The 5694-bp cDNA has a 3312-bp open reading frame, which codes for a 1104 amino acid putative protein with seven transmembrane domains. The predicted protein revealed significant homology with the transient receptor potential (trp) family of Ca(2+) channel proteins. Northern blot analysis indicated that trp-p8 expression within normal human tissues is mostly restricted to prostate epithelial cells. In situ hybridization analysis showed that trp-p8 mRNA expression was at moderate levels in normal prostate tissue and appears to be elevated in prostate cancer. Notably, trp-p8 mRNA was also expressed in a number of nonprostatic primary tumors of breast, colon, lung, and skin origin, whereas transcripts encoding trp-p8 were hardly detected or not detected in the corresponding normal human tissues.

Determination of the glial fibrillary acidic protein in human cerebrospinal fluid and in cyst fluid of brain tumors.

The glial fibrillary acidic protein (GFAP) have been quantitatively determined in over 200 samples of liquid content of brain tumours and in cerebrospinal fluid (CSF) of cases with various tumours of the cerebral nervous system. For establishing the GFAP value, the rocket radioimmunoelectrophoresis was used. The studies were performed in three series of patients. The GFAP value of fluids from 26 cysts of both neoplastic and non-neoplastic type had a wide range of 0.6 microgram/ml to 40 micrograms/ml. Significant elevation of GFAP was usually recorded in fluid from cysts of anaplastic tumour with astroglial differentiation. In this series of 24 cases with various brain tumours, the GFAP value of the CSF ranged from 0.2 microgram/ml to 50 micrograms/ml. In gliomas, as in astrocytoma and glioblastoma, these values were on a higher level, of over 4 micrograms/ml. In other tumours and in cerebral lesions of other aetiology, the GFAP values were lower, below 3 micrograms/ml and 0.3 microgram/ml respectively. In another series of 32 patients with brain tumour treated surgically, a significant increase of GFAP (to 30 micrograms/ml) was noted in the CSF during the first week after operation, and that was always associated with an increase of the total protein of the CSF. During the second and third week after operation, when the total protein of the CSF was reduced to a normal level, the values of GFAP were still elevated, first of all in those cases of astrocytoma and glioblastoma which were not radically excised. These findings suggest that investigation of GFAP in the CSF of patients with brain tumour may be helpful in diagnosis and prognosis.

Medulloblastoma: histological evaluation and prognosis. A clinical, immunohistochemical and electron microscopic study.

Eighty-four cases of medulloblastoma were examined immunohistochemically and 12 by electron microscopy to assess differentiation in these tumors. Based upon the largest series of medulloblastoma studied to date, we demonstrated glial fibrillary acidic protein (GFAP) positivity, in 25% (21/84) of these tumors showing glial differentiation. GFAP-positive cells were seen more frequently in the desmoplastic variant of medulloblastoma (7/10). Under electron microscopy, the major part of the 12 tumors studied appeared primitive and undifferentiated. In 7 cases, groups of cells were found with primitive neuronal and/or glial features. GFAP positivity was confirmed at light microscopy level in all cases where cells showed glial differentiation in the form of glial-like filaments in cytoplasma. However, a follow-up questionnaire study of those patients who had received only surgical treatment revealed no difference in mean survival time between GFAP-positive and GFAP-negative medulloblastoma.

Counterflow affinity isotachophoresis on cellulose acetate membranes.

Counterflow isotachophoresis on cellulose acetate membranes of human alpha-fetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis. Smaller amounts of lectins and more diluted samples of AFP could be used in the described method.

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide:MHC class II complexes in B cells.

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-Ab and an antigenic peptide Ealpha52-68 covalently linked to its beta-chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, Ealpha52-68:I-Ab complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.

Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway.

Presentation of the Mtv-1 superantigen (vSag1) to specific Vbeta-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II alphabeta dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.

Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments.

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse-chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I-Ab with a 17-amino acid peptide derived from the alpha chain of HLA-DR (DR alpha52-68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II-enriched compartments before reaching the cell surface.