Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

Abstract

SummaryThe T cell co-receptor, CD8, binds to the 0 domain of HLA class I (Salter, R .D., R .J. Benjamin, P.K . Wesley, S .E. Buxton, T.P.J . Garrett, C. Clayberger, A.M . Krensky, A .M . Norman, D.R. Littman, and P Parham . 1990. Nature (Lond]. 345 :41; Connolly, J.M ., TA. Potter, E.M. Wormstall, and TH . Hansen. 1988 . J. ExpL Med. 168 :325 ; and Potter, TA., TV. Rajan, R.F. Dick II, and J .A . Bluestone . 1989 . Natur e (Lond.j. 337 :73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (1g)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I . Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the BenceJones homodimer, REI . Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 a-terminal domain and an Ig variable domain.T he TCR-CD3 complex associates with CD8 or CD4 to form a multimeric complex interacting with peptide and HLA class I or HLA class II, respectively, during T cell activation . In addition to their function as adhesion molecules, CD8 and CD4 also serve as signalling molecules. The cytoplasmic domains of CD4 and CDSa bind to the NH2-terminal domain of the intracellular tyrosine protein kinase p56kk (1-3) . Antibody crosslinking of CD8 and CD4 to the TCR-CD3 complex leads to enhanced calcium flux and phosphorylation of a set of proteins, which includes the r chain of the TCR complex (4-6) . The ability of CD8 to induce these changes is dependent on its ability to associate with p56kk . CD8 and CD4 are also important for positive and negative selection in the thymus (7, 8). TCR specificity in T cell repertoire selection is determined by both the TCRa/(3 heterodimer and CD4 or CD8 accessory molecules (9, 10) .The human CD8 molecule is expressed either as an al a homodimer or as an a/(3 heterodimer. The homodimer is expressed exclusively on subsets of TCR-y/S cells and NK cells, and is co-expressed with CD80 on peripheral T cells and thymocytes . Individual human peripheral T cells can express varying amounts of CD8 a/a and alai complexes, and these appear to be differentially regulated upon T cell activation (11). CD8 expression can be induced on human CD4 cells upon activation with mitogens (12) and on murin...

Description of an Ectothermic TCR Coreceptor, CD8α, in Rainbow Trout

“…Human CD8␣ lysine (58) within the CDR2 loop is critical for the electrostatic-based association with the ␣3 acidic loop of HLA. Mutational analysis of the human CD8␣ CDRs resulted in Ͼ70 -80% (CDR1) and 50 -65% (CDR2) reduction in MHC class I binding (6). Moreover, introduction of negatively charged residues within CDR1 abrogated HLA binding, presumably via electrostatic repulsion with the HLA ␣3 acidic loop.…”

“…Base pairs refer to positions within Onmy-CD8␣ cDNA (AF178053). Two sense primers (E1S, 5Ј-GAGCTT GAACGTGTTGCTGT-3Ј; bp [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and nested ES2, 5Ј-AGAGGGTG GAGATCACTTGT-3Ј; bp 126 -145) based on a putative V region cDNA were used in conjunction with an anchored T7 primer (3Ј region of the pCMV-Zap Express MCS) to amplify (30 cycles consisting of 94°C for 10 s, 55°C for 30 s, and 72°C for 1 min) the full-length cDNAs from the trout thymocyte cDNA library. Products were cloned into pBlunt (Invitrogen) and sequenced.…”

“…The ability of CD8 to act as a TCR coreceptor lies in its capacity to interact with MHC class I and ␤ 2 -microglobulin (␤ 2 m) during TCR-mediated MHC peptide recognition (5)(6)(7)(8). Indeed, CD8␣ associates with ␤ 2 m and the ␣2 and ␣3 domains of MHC class Ia molecules using its A/B ␤ strands and the complementary-determining regions (CDR) within the extracellular IgSf V domain.…”

Spondylarthritis in HLA–B27/human β2‐microglobulin–transgenic rats is not prevented by lack of CD8

“…Subsequent analysis of genomic DNA from wild-type and homozygous mutant rats confirmed a T3 C transition that encoded a substitution of a proline residue (CCA) for a highly conserved serine residue (TCA) at amino acid position 94, which is adjacent to the distal cysteine residue that forms the single disulfide loop (7) (Figure 1). This mutation would be expected to profoundly disrupt the formation of the disulfide loop and the folding of the CD8␣ chain, since Pro cannot assume the bond angles that Ser assumes in accommodating disulfide bond formation by the adjacent Cys 93 and since this substitution is predicted to cause the loss of an H-bond between ␤ strands C and F (23,24). A much more conservative missense mutation (Gly3 Ser) in the human CD8A gene at the position 5 residues N-terminal of this Ser was reported to produce the absence of CD8␣ expression, leading to immunodeficiency in a homozygous subject (25).…”

Genomic Organization of the Chicken CD8 Locus Reveals a Novel Family of Immunoreceptor Genes

“…Both the CD8 ␣-and ␤-chains are composed of a single extracellular Ig superfamily (IgSF) 3 variable (V) domain, a membrane-proximal hinge (H) region, a transmembrane domain (TM), and a cytoplasmic tail (CY). Biochemical and structural studies have shown that the Ig V domain of CD8␣, in conjunction with that of CD8␤, interacts with MHC class I molecules, thereby allowing the CD8 molecule to function as a coreceptor of the T cell Ag receptor (2)(3)(4)(5)(6)(7).…”