Peter Cresswell

Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids.

Regulation of HLA class I and class II antigen expression was studied in hybrids of human T and B lymphoblastoid cell lines (LCL). The T-LCL CEMR.3 expresses no HLA class II antigens. It expresses little total HLA class I antigen and no HLA-B antigens. The B-LCL 721.174 is a radiation-induced variant immunoselected for loss of class II antigen expression. In addition to showing a deletion of all HLA-DR and DQ structural genes, 721.174 expresses no HLA-B antigens and a decreased level of HLA-A antigen compared with the parental cell line. A hybrid of 721.174 and CEMR.3 expresses class II antigens encoded by CEMR.3. Increased expression of HLA class I antigens encoded by both 721.174 and CEMR.3 was also observed. Specifically, the previously undetectable HLA-B5 and HLA-Bw6 antigens encoded by 721.174 and CEMR.3, respectively, were present on the hybrid. Increased expression of the HLA-A2 antigen encoded by 721.174 was also observed. An immunoselected variant of the hybrid lacking both CEMR.3-derived copies of chromosome 6 lost expression of the HLA-B5 antigen encoded by 721.174 and expressed a decreased amount of HLA-A2. From these data, we infer that two complementary trans-acting factors mediate enhanced expression of HLA class I antigens in the hybrid. One of these factors is provided by a gene located on chromosome 6, derived from CEMR.3. The second factor, introduced by 721.174, is the gene previously postulated to induce expression of CEMR.3-encoded class I antigens in hybrids of CEMR.3 with B-LCL.

Differential transport requirements of HLA and H-2 class I glycoproteins

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line × T-lymphoblastoid cell line (B-LCL × T-LCL) hybrid, 174 × CEM. T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or 137 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2Dpor H-2Kb class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.

HLA-A2 as a target for cell-mediated lympholysis: Evidence from immunoselected HLA-A2 negative mutant cell lines

Cloned mutants of the human B lymphoblastoid cell line SB have been isolated using mutagenesis with ethyl methanesulfonate followed by negative selection with an anti-HLA-A2 serum and complement. Absorption analysis with 125I Staphylococcus aureus protein A binding to antibody sensitized cells. HLA typing, and immune precipitation analysis showed the mutants to be serologically identical to the SB parent except for the loss of HLA-A2. When tested as target cells for cell-mediated lympholysis by cytotoxic T lymphocytes generated in the mixed lymphocyte response, the SB and mutant cell lines demonstrated comparable susceptibility when the putative targets were HLA antigens other than HLA-A2. However, when compared for susceptibility to lysis by cytotoxic T lymphocytes considered to be HLA-A2 specific, the SB parent was effectively killed whereas little or no killing of the HLA-A2 mutants was observed. The results provide a new line of evidence that HLA antigens recognized by antibody can also be the true molecular targets for cytotoxic T lymphocytes.

Transferrin receptors on human B and T lymphosblastoid cell lines

Experiments demonstrating the existence of receptors for iron-saturated transferrin on both B and T lymphoblastoid cell lines of human origin are described. Binding of 125I-labeled transferrin is rapid, saturable and reversible. It can be specifically inhibited by unlabeled transferrin but not by other proteins. The number of receptors on T cell lines determined by Scatchard analysis is almost double the number on B cell lines but the binding affinities are equal. The putative transferrin receptor can be removed from the cell by the proteolytic enzymes papain and trypsin, and is re-expressed during overnight incubation at 37 degrees C. Resynthesis is inhibited by puromycin. The receptor can be solubilized by deoxycholate, and retains transferrin binding capacity when non-covalently attached to an amphipathic matrix consisting of deoxycholate-coupled poly(L-lysyl) Agarose.

Analysis of monoclonal antibodies reactive with human class II beta chains by two-dimensional electrophoresis and western blotting☆

We have used the Western blotting technique to examine B lymphoblastoid cell line (B-LCL) membrane proteins separated by two-dimensional gel electrophoresis specifically to analyze the binding patterns of monoclonal antibodies to separated HLA class II antigen beta (beta) subunits. The B-LCL LG-10 (homozygous for DR7), in which at least two sets of class II molecules can be distinguished on the basis of different electrophoretic mobilities, was examined with five monoclonal antibodies which detect monomorphic determinants. Four of the antibodies reacted with only DR beta subunits, while one antibody, XD5.A11, reacted with DR and with additional beta chains. Examination of two polymorphic monoclonal antibodies, SFR3-DR5, specific for HLA-DR5, and SFR3-PI.1, which reacts with a determinant absent from DR3 and DR7 homozygous lines, showed that both bind beta subunits from Swei, a DR5 homozygous line. Purification of a subpopulation of Swei class II molecules using an SFR3-PI.1 affinity column showed that the determinants recognized by SFR3-DR5, SFR3-PI.1, and a monomorphic monoclonal antibody reactive only with HLA-DR beta subunits of LG-10, reacted with identical beta subunits. Additional class II antigen subunits reactive with XD5.A11 were nonreactive with the polymorphic antibodies and the HLA-DR-specific monomorphic monoclonal antibody.

An epitope common to HLA class I and class II antigens, Ig light chains, and β2-microglobulin

The homology of class I major histocompatibility complex (MHC) antigens, class II MHC antigens, and immunoglobulin molecules has suggested their divergence from a common ancestral gene. We report here a monoclonal antibody (mAb), PAC. M1, which reacts with HLA class I heavy chains, HLA class II α and β chains, and the light chain of human immunoglobulin by Western blot analysis. PAC.M1 reacted with 44 kd, 33 kd, and 29 kd species when tested on membrane glycoproteins from TRa1, a B-lymphoblastoid cell line (B-LCL). Two-dimensional electrophoresis and Western blotting of TRa1 glycoproteins showed that these species had the appropriate electrophoretic mobilities for class I heavy chain and class II α and β subunits. The presence of the epitope was verified on class II α and β subunits by Western blotting of purified αβ-invariant chain complexes, and on class I heavy chains by Western blotting of purified class I antigens. The PAC. M1 mAb also reacted with immunoglobulin light chains when Western blotting was performed with normal human serum and purified IgG and IgM as antigens. While reactivity of the mAb with beta-2 microglobulin (β2m) was difficult to detect by Western blotting, binding of PAC.M1 to purified β2m was detectable in a solid-phase binding assay. Thus, PAC.Ml reacts with a determinant shared by a number of members of the immunoglobulin superfamily.

A public HLA antigen associated with HLA-A9, Aw32, and Bw4.

TheHLA complex codes for three distinct 44000 dalton molecules associated withΒ2 microglobulin — HLA-A, B and C —each with its own multiallelic series of private antigens. The HLA-B molecule is exceptional in that it also carries a diallelic system,Bw4 andBw6. One of these,Bw4, is often associated with the A- locus specificity A9. This finding has usually been ascribed to linkage disequilibrium between A-and B-locus antigens. We have shown, however, that an epitope called LHe is actually shared by A-locus and B-locus molecules. This epitope is found on all HLA-B molecules bearing the Bw4 determinant and is also found on all HLA-A molecules carrying the A9 (Aw23 and Aw24) or Aw32 specificities. We consider this a “public” HLA antigen; the possible molecular basis for both subtypic and public antigens on a single glycoprotein is discussed.

Robbit antisera to human B cell alloantigens: Effects on the mixed lymphocyte response☆

Abstract Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes. Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′) 2 or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the α1 and α2 domains. Human (HLA-B7) and mouse (H-2Dp) hybrid class I genes, encoding the leader, α1, and α2 sequences of one species fused to the α3, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, α1, and α2-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, α1, and α2 domains fused to the mouse α3, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the α1 and/or α2 domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules.

HLA-DR7-Specific monoclonal antibodies and a chimpanzee anti-DR7 serum detect different epitopes on the same molecule

We describe here two monoclonal antibodies with HLA-DR7 serologic specificity. The antibodies, SFR16-DR7M, a cytotoxic rat IgM antibody of high affinity, and SFR16-DR7G, a noncytotoxic antibody of the rat IgG 2a class, react with only DR7-positive cells in radioimmunoassay. The cytotoxic activity of SFR16-DR7M correlates completely with the presence of the DR7 specificity, and segregates with the DR7-bearing haplotype in a family. SFR16-DR7M precipitates a class II molecule with the electrophoretic characteristics of DR molecules from LG-10, an HLA-DR7 homozygous cell line. SFR16-DR7G completely inhibits the cytotoxicity of SFR16-DR7M, but only partially inhibits the cytotoxicity of a chimpanzee antiserum with DR7 specificity, Gay/Swei. In binding-inhibition studies, binding of SFR16-DR7M to LG-10 cells is only partially inhibited by the chimpanzee antiserum and vice versa. Both SFR16-DR7M and Gay/Swei reciprocally deplete the same class II molecules from a 35S-methionine-labeled detergent-solubilized membrane preparation of the LG-10 cell line. The chimpanzee serum Gay contains antibodies reactive with epitopes on separated DR7 beta chains, while both SFR16-DR7M and SFR16-DR7G bind only to DR7 alpha-beta complexes. These data suggest that at least two allogeneic epitopes exist which result in the same serologic specificity, and that these epitopes differ in their requirement for alpha-beta complex formation.