Development and characterization of human constitutive proteasome and immunoproteasome subunit‐specific monoclonal antibodies

Abstract

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

HLA class I antigen and transporter associated with antigen processing downregulation in metastatic lesions of head and neck squamous cell carcinoma as a marker of poor prognosis

“…Immunoperoxidase staining of formalin-fixed, paraffin-embedded tissue sections with mAb was performed utilizing the EnVision+ system (Dako Cytomation, Carpinteria, CA, USA) as described (26). The percentage of stained tumor cells in each lesion was evaluated independently by two investigators.…”

Melanoma immunotherapy using mature DCs expressing the constitutive proteasome

“…Proteasome subunit-specific mAbs were provided by S. Ferrone (Roswell Park Cancer Institute, Buffalo, New York, USA) and used as previously described (29). Briefly, DCs were fixed with 2% paraformaldehyde, microwave treated, and permeabilized with 0.1% saponin.…”

Alterations in the antigen processing-presenting machinery of transformed plasma cells are associated with reduced recognition by CD8+ T cells and characterize the progression of MGUS to multiple myeloma

“…The following antibodies were produced and characterized as described previously [18][19][20][21][22] : the ␦-specific monoclonal antibody (mAb) SY-5; the MB1-specific mAb SJJ-3; the -specific mAb NB1; the LMP2-specific mAb SY-1; the LMP7-specific mAb HB2; the LMP10-specific mAb TO-7; the TAP1-specific mAb NOB1; the TAP2-specific mAb NOB2; the calnexin-specific mAb TO-5; the calreticulin-specific mAb TO-11; the ERp57-specific mAb TO-2; the tapasin-specific mAb TO-3; and the ␤ 2 -m-specific mAb NAMB-1. They were labeled with fluorescein isothiocyanate (FITC) by the use of the Pierce FITC Antibody Labeling Kit.…”