ABSTRACT
The identification of all CD28/CTLA-4 counterreceptors is critical to our understanding of this pivotal pathway of T cell activation. Clouding our understanding has been the reported discrepancies in expression and function of the B7-1 (CD80) molecule based upon the use of the BB1 vs other anti-B7-1 mAbs. To resolve this issue, we have cloned a BB1-binding molecule from the BB1+B7-1(-) NALM-6 pre-B cell line. Here, we demonstrate that this BB1-binding molecule is identical to the cell surface form of CD74 (MHC class II-associated invariant chain). CD74-transfected cells bound the BB1 mAb but not other anti-CD80 mAbs, CD28-Ig, or CTLA4Ig. Absorption and blocking experiments confirmed the reactivity of BB1 mAb with CD74. A region of weak homology was identified between CD74 and the region of B7-1 encoding the BB1 epitope. Therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Many of the puzzling observations in the literature concerning the expression of human B7-1 are resolved by an understanding that BB1 staining is the summation of CD74 plus B7-1 expression. This observation requires the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.
Subject(s)
Antibodies, Monoclonal/metabolism , Antigens, Differentiation, B-Lymphocyte/immunology , Antigens, Differentiation/metabolism , B7-1 Antigen/immunology , CD28 Antigens/metabolism , Histocompatibility Antigens Class II/immunology , Immunoconjugates , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , 3T3 Cells , Abatacept , Amino Acid Sequence , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Antigens, CD , Antigens, Differentiation/immunology , Antigens, Differentiation, B-Lymphocyte/genetics , B7-1 Antigen/biosynthesis , Binding Sites, Antibody/immunology , CD28 Antigens/immunology , CHO Cells , COS Cells , CTLA-4 Antigen , Cloning, Molecular , Cricetinae , Histocompatibility Antigens Class II/genetics , Humans , Immunoglobulin Fc Fragments/metabolism , Interferon-gamma/pharmacology , Keratinocytes/metabolism , Mice , Molecular Sequence Data , Phenotype , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16-subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRalpha/beta+CD4-CD8+CD11b+CD28-CD45RO+C D56-CD101+CD103+ (alphaEbeta7 integrin). In addition, BY55 was expressed on most CD8+CD28- peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.
Subject(s)
Antigens, CD , Immunoglobulins/genetics , Intestinal Mucosa/immunology , Killer Cells, Natural/metabolism , Lymphocyte Subsets/metabolism , Membrane Proteins/genetics , Multigene Family/immunology , Receptors, Immunologic , T-Lymphocytes, Cytotoxic/metabolism , Amino Acid Sequence , Animals , CD28 Antigens/blood , CD56 Antigen/biosynthesis , CD56 Antigen/blood , CD8 Antigens/blood , Cloning, Molecular , GPI-Linked Proteins , Glycosylphosphatidylinositols/metabolism , Humans , Immunoglobulins/chemistry , Intestinal Mucosa/cytology , Intestinal Mucosa/metabolism , Lymphocyte Activation , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/immunology , Mice , Molecular Sequence Data , Precipitin Tests , RNA, Messenger/biosynthesisABSTRACT
The functional necessity for two CD28 counterreceptors (B7-1 and B7-2) is presently unknown. B7-1 and B7-2 equivalently costimulate IL-2 and interferon-gamma (IFN gamma) production and IL-2 receptor alpha and gamma chain expression. B7-2 induces significantly more IL-4 production than B7-1, with the greatest difference seen in naive T cells. Repetitive costimulation of CD4+ CD45RA+ T cells with B7-2 results in moderate levels of both IL-4 and IL-2, whereas repetitive costimulation with B7-1 results in high levels of IL-2 and low levels of IL-4. Therefore, B7-1 and B7-2 costimulation mediate distinct outcomes, since B7-2 provides an initial signal to induce naive T cells to become IL-4 producers, thereby directing the immune response more towards Th0/Th2, whereas B7-1 is a more neutral differentiative signal.
