Location of the epitope for an anti-CD8alpha antibody 53.6.7 which enhances CD8alpha-MHC class I interaction indicates antibody stabilization of a higher affinity CD8 conformation

MHC class I tetramers are widely used, usually in combination with an antibody to CD8, to detect antigen specific T cells. Some anti-CD8alpha antibodies block the interaction of murine MHC class I tetramers with CD8 T cells, while others such as 53.6.7, enhance. To understand the molecular basis for this effect, we mapped the epitope for the enhancing antibody 53.6.7 and three other blocking antibodies using a panel of murine CD8alpha (Lyt-2) mutants expressed on COS-7 transfectants. Mutations in residues that contact MHC class I affected binding of the blocking antibodies. In contrast, antibody 53.6.7 was affected by a mutation in the residue T81A located on the D-E loop. In the cocrystal of CD8alphaalpha with MHC class I, two different complexes (A and B) were observed, indicating the existence of different CD8 conformations. The T81 residue does not make contact with MHC class I in either complex, however, neighboring residues in the D-E loop make very different contacts in the two different complexes. The most likely explanation for antibody enhancement of tetramer bindings is that binding of 53.6.7 to CD8alphabeta stabilizes a conformation with a higher affinity for interaction with MHC class I and suggests that the CD8 binding site is flexible.     

A feline CD2 homologue interacts with human red blood cells

A cDNA encoding a feline homologue of CD2 (fCD2) was identified. Several amino acids (aa) important for ligand interaction, molecular folding or signal transduction, found in other mammalian CD2, were found to be highly conserved in the predicted fCD2 aa sequence. fCD2-expressing cells were able to form rosettes with human red blood cells (probably via human CD58), and the rosette formation was inhibited by an anti-fCD2 monoclonal antibody. These results are indicative of the similarity of feline and human CD2 structures. fCD2 was found to be expressed in feline peripheral blood T lymphocytes, monocytes and cultured lymphoid cells.     

Monoclonal antibody against murine T cell receptor V alpha 14 cross-reacts with human CD3 epsilon and detects disulfide-linked dimeric form.

A monoclonal antibody against V14+ alpha-chain of murine T cell receptor (TCR) was established by fusing spleen cells from a rat immunized with a soluble chimeric TCR/IgG3 protein containing murine TCR V alpha 14J alpha 281 in place of the VHDHJH of an IgG3. lambda 1, and subjected to screening on a human transfectant (Jurkat variant) expressing the murine V14J281 alpha-chain. The anti-mouse V alpha 14 antibody precipitated TCR alpha beta molecules from Triton X-100-solubilized extracts of 125I-labeled murine thymocytes and spleen cells. Unexpectedly, the antibody showed cross-reactivity to the human CD3 epsilon molecule and detected a disulfide-linked 20 kDa dimeric form of human CD3 epsilon, which is a novel family component of the CD3 complex and is associated closely with the CD3 zeta-zeta homodimer as well as TCR alpha beta or TCR gamma delta.     

The cytoplasmic domain of the CD8 alpha-chain is required for its interaction with p56lck

The CD8 glycoprotein expressed on the surface of CTLs is a heterodimer composed of alpha (Lyt-2) and beta (Lyt-3) chains. Recent studies have shown that CD4 and CD8 are physically associated with a T cell-specific protein-tyrosine kinase p56lck. Our previous experiments have suggested strongly that p56lck interacts directly with CD4 and CD8 molecules. The present report using cytoplasmic deletion mutants of the CD8 alpha-chain gene has extended our observations to demonstrate unequivocally that the cytoplasmic domain of the CD8 alpha chain is responsible for interaction with p56lck. The data has also confirmed the importance of the conserved twelve amino acid sequence motif of the CD8 alpha cytoplasmic domain in complex formation with p56lck.     

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.     

A cDNA encoding feline CD4 has a unique repeat sequence downstream of the V-like region.

We have cloned, sequenced and expressed a cDNA encoding the feline CD4 glycoprotein. This clone, termed FT121, contains a unique repeat sequence downstream of the V-like region which has not been reported in other mammalian CD4 cDNA. In addition, the cysteine residues at positions 45 and 175 have an unusual pattern. On the other hand, regions other than the unique sequence share significant homology with human CD4 and mouse L3T4 cDNA in the amino acid sequence deduced from the nucleotide sequence, especially in the putative cytoplasmic region. Here, we show that FT121 has a complete open reading frame and efficiently expresses feline CD4 molecules on COS cells. Further we found that the cytoplasmic components, lymphocyte-specific protein-tyrosine kinase p56lck binding sites and phosphorylation site (serine resides), are well conserved, though the extracellular region of the feline CD4 molecule deduced from FT121 is probably different from that of human CD4 and mouse L3T4.     

Molecular cloning of the feline CD8 beta-chain.

Human mouse and rat CD8 have been described as being disulphide-linked heterodimers of alpha and beta chains. More recently the chicken alpha and beta chains were described. In the bovine and feline immune system only the z-chain was reported. In this study we have cloned and determined the nucleotide sequence of a cDNA encoding the beta-chain of the feline T-cell surface antigen CD8. Using a nested polymerase chain reaction- (PCR) and two primer pairs designed from the human CD8 beta cDNA nucleotide sequence, we amplified a 430 base pair fragment from a feline thymus cDNA library which was used as a probe for screening the feline library at high stringency. After three rounds of screening, five clones were isolated. A clone, named FTb-6, containing a 3.8 kilobase pair insert was mapped, sequenced and compared with the published sequences of the genes encoding the human, mouse, rat and avian CD8 beta. We have determined the primary structure of the feline CD8 beta. The feline CD8 beta has an open reading frame, 630 nucleotides in length encoding a protein with 210 amino acid residues and its composition showed that the feline molecule is a member of the immunoglobulin gene super family.     

A murine anti-sheep T8 monoclonal antibody, ST-8, that defines the cytotoxic T lymphocyte population

A mouse monoclonal antibody (mAb), ST-8, reactive with a subpopulation of sheep T lymphocytes was investigated by tissue distribution analysis and functional studies of the antigen-bearing (ST-8+) cells. Two other mouse mAb, ST-1 and T-80 that recognize all sheep T cells and a T-cell subset, respectively, were also examined for comparison. The ST-8+ cells represented 61-69% of thymocytes and about 10-30% of peripheral T lymphocytes. Histologically, ST-8+ cells were found mainly in the thymic cortex, T-dependent areas of the peripheral lymphoid tissues and the splenic red pulp and marginal zone, but not in B-dependent areas such as germinal centers of lymph follicles of the Peyer's patches. ST-8 mAb plus complement treatment completely abolished both the induction phase and the effector phase of alloreactive cytotoxic T lymphocytes but did not affect proliferative responses to T-dependent mitogens and allogeneic antigens. ST-8 mAb also blocked the cytotoxic T lymphocyte function of lysing specific targets in the absence of complement. ST-8 mAb immunoprecipitated an antigen from the surface of sheep T lymphocytes under reducing conditions with an apparent molecular weight of 33-35 kilodaltons. Therefore the antigen recognized by the ST-8 mAb showed striking similarities to human T8/Leu-2a and mouse Lyt-2 antigens not only in the tissue distribution and function of antigen-bearing cells but also the molecular weight. We conclude that the ST-8 antigen is the ovine homologue of the human T8 and mouse Lyt-2 antigens.     

Generation of anti-human CD8 beta-specific antibodies using transfectants expressing mixed-species CD8 heterodimers

The CD8 glycoprotein is a lymphocyte differentiation antigen comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. The majority of monoclonal antibodies which recognize the human CD8 antigen react with the CD8 alpha chain, while a single mAb, referred to as T8/2T8-5H7 (or 2ST8-5H7), has been identified which binds to the CD8 alpha/beta heterodimer. In order to generate antibodies specific for CD8 beta, murine fibroblast transfectants were constructed which express the human CD8 beta chain in combination with either the human CD8 alpha chain or the murine CD8 alpha homologue, the Lyt-2 molecule. These transfectants were used to raise polyclonal heteroantisera in mice. Transfectants expressing human CD8 alpha/beta heterodimers induced moderate anti-CD8 alpha titers, but were weakly effective in generating anti-CD8 beta titers, despite high level cell surface expression of this protein. In contrast, transfectants expressing mixed-species CD8 heterodimers (murine CD8 alpha and human CD8 beta) induced high anti-CD8 beta titers in immunized mice. Following fusion of splenocytes from mice immunized with mixed-species CD8 transfectants, the mAb 5F2 was isolated which specifically recognizes the human CD8 beta chain. Unlike T8/2T8-5H7, the mAb 5F2 can bind the CD8 beta chain irrespective of its pairing partner, and can immunoprecipitate the CD8 beta protein from cells transfected with the CD8 beta gene in the absence of the human or mouse CD8 alpha gene product. Anti-CD8 beta antibodies should help elucidate the extent of biochemical heterogeneity of the CD8 beta protein, and will also be useful in defining the role of the CD8 beta protein in thymocyte and lymphocyte development, recognition and activation.     

Characterization of duck leucocytes by monoclonal antibodies

cDNAs encoding CD3epsilon, CD4 and the alpha-chain of CD8 of ducks were cloned. Monoclonal antibodies against Pekin duck CD4 and CD8alpha revealed that the antigens were expressed by the majority of thymocytes and by subpopulations of CD3+ cells in peripheral tissues. CD8alpha cell surface expression was also found on 90% of bursal cells. The B cell specificity of a newly developed mab to the immunoglobulin light-chain (L-chain) was confirmed by double labelling studies with the chicken B-cell cytokine BAFF. Using these tools and a mab reacting with the cytoplasmic domain of CD3epsilon, we demonstrated that the CD8alpha molecule is expressed to high levels in CD3-positive T cells and at lower levels in CD3-negative bursal and peripheral B cells. Mab 2-4, which recognizes the chicken CD28 molecule, was found to react with CD4-positive duck lymphocytes and with CD8-positive duck T cells but not with CD8-positive B cells. Mab K1, which recognizes a common antigen on chicken thrombocytes and monocytes/macrophages, was found to react with duck thrombocytes and macrophages. Thus, a range of mabs is now available which will allow to phenotype the major leucocyte populations in ducks, a surrogate infection model for hepatitis B virus.     

Differential expression and regulation of the human CD8α and CD8β chains

The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The majority of monoclonal antibodies (mAb) reactive with the human CD8 antigen bind the CD8 alpha chain, while a single mAb, T8/2T8-5H7, has been identified which binds to the CD8 alpha/beta heterodimer. While the two chains of CD8 have been presumed to be coordinately expressed in normal T cells, this is not always the case. Northern blot analysis of a panel of T-cell leukemias and normal cells demonstrate that CD8 alpha and CD8 beta are not invariably co-transcribed and phenotypic analysis of fresh and interleukin 2 (IL-2) expanded peripheral blood mononuclear cells (PBMC) confirm that the CD8 alpha and CD8 beta chains are differentially expressed at the cell surface. Four distinct subpopulations of CD8+ cells have been identified based on the expression of CD8 alpha/alpha or CD8 alpha/beta complexes: (1) T-cell receptor (TcR) alpha beta+ T cells which are CD8 alpha+/beta+; (2) TcR alpha beta+ T cells which are CD8 alpha+/beta-; (3) TcR gamma delta+ T cells which are CD8 alpha+/beta- and (4) natural killer (NK) cells which are CD8 alpha+/beta-. We also demonstrate the down-regulation of the CD8 alpha/beta heterodimers from the surface of a CD8+ T-cell clone following treatment with phorbol myristate acetate (PMA) while CD8 alpha/alpha homodimers remain on the cell surface. This observation demonstrates that a) a CD8+ T-cell clone can express both CD8 alpha/alpha homodimers and CD8 alpha/beta heterodimers and b) these two complexes do not have identical biological properties. Together, these data suggest that CD8 alpha/alpha and CD8 alpha/beta dimers may not subserve identical functions. The differential contribution of these two CD8 complexes should be considered in models of T-cell-mediated cytotoxicity and T-cell activation.     

CD3+4-8-WT31-(T cell receptor gamma+) cells and other unusual phenotypes are frequently detected among spontaneously interleukin 2-responsive T lymphocytes present in the joint fluid in juvenile rheumatoid arthritis. A clonal analysis

T lymphocytes (E rosetting cells) isolated from the joint fluid of four patients with juvenile rheumatoid arthritis (JRA) were first analyzed for surface antigen expression. Approximately 15% of cells were CD25+ (interleukin, IL, 2 receptor positive), in addition, a remarkable proportion of cells expressed the CD2+3- phenotype. CD3+ cells outnumbered the sum of CD4+ and CD8+ cells as well as the cells reactive with the WT31 monoclonal antibody (which recognizes a framework determinant of the alpha/beta T cell receptor). Purified T cells were cloned under culture conditions (1% phytohemagglutinin, PHA plus IL2) which allow clonal expansion of most peripheral blood T lymphocytes. Under these conditions proliferating cells ranged from 25 to 65%; clones (derived from microcultures containing 0.5 or 0.25 cells/well) were tested for cytolytic activity against P815 cells (in the presence of PHA) or against the natural killer (NK)-sensitive K562 target cells. Fifty-four percent and 73% of clones obtained from the two patients with the polyarticular form of the disease displayed cytolytic activity in the lectin-dependent assay. Cytolytic clones were 22 and 29% in the two patients with single joint involvement. About half of all cytolytic clones displayed NK-like activity. Surface antigen analysis revealed that, in addition to conventional CD3+4+8- and CD3+4-8+, a noticeable fraction of clones (50/202) displayed unusual surface phenotypes. In particular, 33/50 coexpressed CD4 and CD8 antigens; 7/50 were CD2+3-4-8- and displayed NK-like activity; 10/50 expressed CD3 but lacked both CD4 and CD8 antigen and did not react with the WT31 monoclonal antibody. In order to allow selective growth of IL2-responsive cells, T lymphocytes were also cloned directly in IL2. As much as 57% of all clones thus obtained (48/84) displayed cytolytic activity. Moreover, about half expressed unusual surface phenotypes including CD2+3-4-8-, CD3+4+8+ and CD3+4-8-WT31-. Given the accumulation at the site of the joint involvement of unusual T cells, most of which displayed cytolytic activity and were likely to represent cells activated in vivo (IL2 responsive), one may speculate that these cells may be involved in the injury process.     

Evidence of cytotoxic T-cell destruction of epidermal cells in human graft-vs-host disease. Immunohistology with monoclonal antibody TIA-1.

A newly described mouse monoclonal IgG1 antibody, TIA-1, binds serine esterase-positive granule membranes of cytotoxic human T cells and is a candidate for an effector molecule involved in T-cell cytolytic mechanisms analogous to those of the perforin system. We performed immunohistologic studies on frozen human skin (n = 5) and lip (n = 21) sections as well as on control frozen sections of tonsils, purified cytolytic T (CD8) cells, and B cells using an indirect immunoperoxidase system. We found a strong association of TIA-1+ cells with CD8+ cells invading the epidermis in lip and skin lesions of graft-vs-host disease in human marrow allograft recipients, as well as a sharp geographic association of TIA+ lymphocytes with CD8+ regions in human tonsil sections. Double staining of CD8 and TIA-1 antigens with fluorescein isothiocyanate and Texas red confirmed that 80% to 90% of the CD8 cells were TIA-1+ in the epidermal infiltrates. Leu-7 activity (natural killer cell) was minimal and found in only three of 17 lip biopsy specimens. These data provide new evidence that direct cytolytic attack by donor T lymphocytes is the mechanism of epithelial target cell killing in human graft-vs-host disease.     

Expression of different CD8 isoforms on distinct human lymphocyte subpopulations.

Human CD8+ lymphocyte subpopulations were analyzed for their expression of CD8 alpha and CD8 beta subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural killer and T cell subpopulations demonstrate that CD3- natural killer cells, T cell receptor gamma/delta, and CD4+CD8+ T cell clones express exclusively CD8 alpha gene products. Structural analysis of CD8 molecules demonstrates that CD8 alpha+/beta- T lymphocytes surface express 75-kDa CD8 alpha/alpha homodimers whereas CD8 alpha/beta lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as alpha/alpha homodimers and alpha/beta heterodimers on CD8 alpha/beta+ cells. Importantly, we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 alpha/beta+/CD8 alpha/alpha+ T lymphocytes, cytolytic activity of CD8 alpha/beta-/CD8 alpha/alpha+ T cell clones was not inhibited by anti-CD8 monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross-linking.     

The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.     

Identification of a putative cellular receptor for feline immunodeficiency virus as the feline homologue of CD9.

A monoclonal antibody vpg15 has been identified which recognizes a putative cellular receptor for feline immunodeficiency virus (FIV). The antibody immunoprecipitates a single 24,000 MW species from feline cells. The molecular size and pattern of expression of the ligand for the vpg15 antibody displayed similarities to that of the human leucocyte differentiation antigen CD9. The reactivity of the vpg15 antibody was therefore compared with that of the anti-human CD9 antibody FMC56, an antibody which cross-reacts with feline cells. Expression of the vpg15 ligand correlated well with the reactivity of the FMC56 antibody on peripheral blood leucocytes and a panel of feline cell lines. Furthermore, the anti-human CD9 antibody reacted with murine fibroblast cells which had been transfected with high molecular weight feline DNA and immunoselected with the vpg15 antibody. FMC56 and vpg15 immunoprecipitated a similar 24,000 MW species from surface-iodinated feline cells and depletion of the vpg15 ligand from cell lysates resulted in a corresponding depletion of the FMC56 ligand. The data demonstrate that the vpg15 antibody recognizes the feline homologue of human CD9 and implicate feline CD9 as a cellular receptor for FIV.     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.