The natural abundance of lambda2-light chains in inbred mice.

The amino acid sequence of the constant (C) domain of the light chain of the mouse myeloma protein M315 has not been identified so far in any other myeloma protein. In this study, serological analysis with antiserum to the C-domain of this light chain (L315) showed that approximately equal to 1% of Igs in normal mouse serum have L chains of the L315 type (called lambda2). Corroborative evidence was obtained by analysis of the carboxyterminal amino acid removed from normal light chains by carboxypeptidase A. A survey of 35 inbred mouse strains showed that all had lambda2; the serum level of Igs with lambda2-chains ranged from approximately equal to 140 microgram/ml in AL/N mice to approximately equal to 25 microgram/ml in SJL, BSVS, and eight other strains. In accord with the anti-Dnp activity of M315, sera from mice immunized with Dnp-KLH had three- to fivefold more lambda2 than sera from control mice immunized with KLH. It was also possible to measure serum immunoglobulin molecules bearing the lambda2 variable region of M315 (VL315). In BALB/c sera, the concentration of VL315 was about sixfold lower than that measured for lambda2. Thus, lambda2-chains are divided into at least two subsets: those whose V domain is indistinguishable from VL315 and those whose VL differs from VL315. A 10-fold increase in VL315 was obtained by immunizing BALB/c mice with Dnp-KLH. The relationship of the VL domains of normal immunoglobulin lambda2-chains to the embryonic Vlambda gene recently sequenced by Tonegawa et al., is discussed.

HLA class II regulation and structure. Analysis with HLA-DR3 and HLA-DP point mutants.

Point mutations that affect HLA-DR structure or expression have not previously been described. In the present study, we isolated such mutants by immunoselection of an ethyl methanesulfonate-mutagenized HLA-DR3 cell line with an anti-HLA-DR3 monoclonal antibody, 16.23. To facilitate analysis, we used a parent cell line with a preexisting deletion of one haplotype encompassing DR and DQ alpha and beta. The selection yielded two sets of mutants, one with defects in DR3 structure, the other with defects in different steps leading to DR expression. Of the expression-defective mutants, one had undergone a second deletion removing the remaining DR alpha gene but no other class II genes. It had a normal abundance of DR beta mRNA but had lost binding of DR monomorphic antibodies, indicating that DR beta chains do not form noncognate dimers. A second mutant had an abnormally large DR alpha mRNA, probably resulting from a splice site mutation. Several mutants had marked reductions in DR beta mRNA levels; in two of these, the lesion appeared to be transcriptional because the reduction in DR beta mRNA was paralleled by an altered methylation pattern of one of the DR beta genes. Other expression-defective mutants had different posttranscriptional defects. Some of the mutations were similar to those that have been found in mouse strains defective in I-E expression, whereas others have no known natural counterpart. The matrix of reactivities of anti-HLA class II monomorphic antibodies with these and similar mutants allowed us to define the gene products recognized by these antibodies. A set of seven mutants were "epitope defective," that is, they expressed normal or near normal levels of HLA-DR3 but no longer bound 16.23. Unexpectedly, each of the epitope mutants had decreased DR dimer stability. These mutants should be useful in localizing the DR3 alloepitope and in elucidating its contribution as a restriction element in the presentation of soluble antigen to immune T cells.

Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content.

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

Variants of HLA-DRw52 defined by T lymphocyte clones.

Polymorphism within the gene encoding the DRw52 allospecificity was studied with DRw52-specific proliferative T lymphocyte clones. Three clones, C6, E3 and ZUK16, were generated by intra-DRw52 priming in mixed lymphocyte culture and tested against an HLA-D homozygous reference cell panel. The reactivity of each clone could be specifically inhibited by anti-DR, but not anti-DQ or anti-DP, monoclonal antibodies. Clone C6 identified a DRw52 variant termed 52a that was predominantly expressed by HLA-B8,DR3+ and DRw13,Dw18+ cells. Clone E3 identified a variant termed 52b which was predominantly expressed by HLA-B18,DR3+,DRw11,Dw5+ and DRw14,Dw9+ cells. Clone ZUK16 identified a variant termed 52c which was predominantly expressed by DRw13,Dw19+ cells. The DRw52a, 52b and 52c variants correspond to the Dw24, Dw25 and Dw26 alleles defined by the WHO HLA 1987 Nomenclature Committee. Together, clones E3 and ZUK16 appeared to identify a fourth DRw52 variant termed 52d which was expressed by two cells, one DR3, Dw"3.3"+ and one DRw14,Dw"9.2"+. A fifth Drw52 variant termed 52e, expressed by a DRw13,Dw"OMW"+ cell, was suggested by the absence of reactivity with any of the three T cell clones. These data thus demonstrate the existence of three well-defined allelic variants of DRw52 and indicate that there are at least two additional variants. The recognition of these polymorphisms by alloreactive T cells provides one measure of their functional significance.

Factors governing the binding and recognition of foreign and self-peptides by MHC class II.

Considerable progress has been made in understanding the basis of T cell recognition and T cell activation. This knowledge has recently been used to modulate T cell activation in animal models of experimental autoimmune disease by two means--selective MHC blockade and peptide-induced tolerance. The use of peptides to interfere with the binding of autoantigenic peptides to MHC requires knowledge of both the class II allele which presents the immunodominant peptide to autoimmune T cells and the identification of peptide analogs that bind with high affinity to that allele. The alternative strategy of peptide-induced tolerance will require identification of the autoantigen and its immunodominant peptides. While the latter approach holds great promise for immunointervention, its wide application will require full knowledge of the mechanisms by which tolerance to self is maintained and how it can be broken.

Unassembled HLA-DR beta monomers are degraded rapidly by a nonlysosomal mechanism.

We previously observed that in a mutant B lymphoblastoid cell line which has a homozygous HLA-DR alpha deletion, DR beta-chains appeared to be unstable. In the present study, we have studied the pathway that leads to degradation of unassembled DR beta-chains. Unassembled DR beta-chains are degraded rapidly in the DR alpha deletion mutant cells, compared with the assembled DR heterodimers present in non-mutant cells. Accelerated DR beta turnover in 9.22.3 cells is specific; class I molecules in these DR alpha-deficient cells turned over slowly. DR beta-chains assemble with Ii in the DR alpha deficient cell line, but this did not protect DR beta-chains from degradation. The maturation of unassembled beta-chains is arrested before their reaching the medial Golgi compartment, and this degradation proceeds by a nonlysosomal, nonendosomal pathway. Degradation of DR beta-chains is blocked when cells are cultured at 16 degrees C, a temperature known to prevent vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus. Degradation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, a drug that is also known to inhibit protein transport from the ER. The results, taken together, suggest that degradation of unassembled DR beta-chains occurs by a nonlysosomal, nonendosomal pathway which involves transport of DR beta-chains out of the ER.

HLA-DR beta chains enter into an aggregated complex containing GRP-78/BiP prior to their degradation by the pre-Golgi degradative pathway.

HLA class II molecules are membrane proteins which are assembled in the endoplasmic reticulum shortly after synthesis of the alpha and beta and invariant chain (Ii) monomers. DR beta chains, in the absence of DR alpha, are rapidly and completely degraded by the pre-Golgi degradative pathway. Here we have examined those factors which target DR beta chains for degradation in a DR alpha deficient cell line, 9.22.3. The DR beta monomers in 9.22.3 were initially incorporated into a proteinaceous complex containing BiP. With time, the DR beta complexes were further aggregated. In wild type cells, which can assemble DR alpha-beta dimers, the secondary phase of aggregation of DR beta was not seen. Additional evidence that aggregation of DR beta in 9.22.3 cells was progressive was that a more mature form of DR beta was found exclusively in the largest DR beta complexes. Furthermore, the most highly aggregated DR beta chains were degraded more rapidly than bulk DR beta chains. These data suggest that DR beta aggregates are intermediates in the pre-Golgi pathway of DR beta degradation. They further suggest that formation of large DR beta aggregates is a proximal event to DR beta degradation. We conclude that DR beta chains are targeted for degradation as a consequence of a change of state, coincident with their aggregation into slow forming, high molecular weight complexes.

Mutations affecting antigen processing impair class II-restricted allorecognition.

Both exogenously derived and endogenously derived Ag generally require processing for their optimal binding and presentation by class I and class II major histocompatibility proteins. It is not known whether steps involved in Ag processing also affect the recognition of alloreactive T cells. We have recently described B cell mutants which have general defects in the processing and presentation of a variety of exogenous Ag to class II restricted T cells. In this report we have studied the ability of these processing mutants to stimulate a set of anti-DR3-specific alloreactive T cells clones. These processing/presentation mutants express normal MHC class II molecules, both in terms of primary sequence and cell surface abundance, but they appear unable to generate effective peptide-MHC complexes. When tested for their ability to stimulate MHC class II alloreactive T cell clones, only one of four T cell clones was stimulated by these mutants; the other three alloreactive T cell clones were not stimulated by either of two different mutants. Both of these mutants express normal levels of the accessory molecules, LFA-3 and ICAM-1. The inability of these mutants to stimulate three of four alloreactive clones indicates that the capacity to be recognized by many alloreactive T cells is linked to the Ag processing capacity of a stimulator cell.

Characterization of a novel form of transferrin receptor preferentially expressed on normal erythroid progenitors and precursors.

A panel of monoclonal antibodies (MoAbs) against cell surface proteins of early BFUe progeny was characterized. Five of these antibodies (Abs) reacted with normal erythroid, but not myeloid, bone marrow cells. Each of the five antibodies, typified by Ab 69.20, immunoprecipitated a dimeric complex of 185,000, which is composed of two identical disulfide-bonded subunits. This antigen had affinity for transferrin, and was essentially identical in biochemical characteristics to transferrin receptors precipitated with the well-characterized MoAbs OKT9 and 5E9. However, this form of transferrin receptor lacked both the OKT9 and 5E9 antigenic determinants and, moreover, the 69.20 epitope was absent from the conventional transferrin receptor, as defined by Abs OKT9 and 5E9. Modulation experiments demonstrated that both 69.20 and OKT9 modulated large, virtually independent populations of transferrin receptors. Both forms of transferrin receptor appeared to be derived from the product of a single gene, but the form defined by MoAb 69.20 apparently predominates in cells of the erythroid lineage and some transformed cell types that manifest a special requirement for iron. These data suggest that cells with a high iron requirement synthesize two forms of transferrin receptor, possibly by means of differential mRNA splicing or by posttranslational modification of the transferrin receptor.

Human T cell proteins recognized by rabbit heteroantisera and monoclonal antibodies.

Many structural and functional features of the cell surface antigens of human T cells remain to be characterized. In these investigations, heteroantisera have been used to identify potentially interesting antigens not yet recognized by individual monoclonal antibody preparations. These antigens include patterns of bands (Mr 140-240K) on various T cells and T cell lines, at least six other bands on T cell lines and novel antigens on CTL lines. The majority of these antigens are different from the compiled list of antigens recognized by monoclonal antibodies against T cell proteins. The monoclonal OKT9 recognizes an antigen most prevalent on dividing cells, and this antigen is the same as that recognized by another monoclonal antibody, 5E9, and by previously described heteroantisera. The monoclonal antibody 4F2 has a distribution similar to OKT9, but it is structurally distinct, and is also present on monocytes. Three T cell surface proteins, two defined by the monoclonal antibodies OKT6 and OKT10, recognize proteins which resemble HLA antigens physiochemically. OKT6 and NAI/34 both recognize a 49,000 glycoprotein which is associated with a 12,000 subunit which reacts with anti-beta 2-microglobulin antibodies. OKT10 recognizes a 46,000 protein, also found with a 12,000 peptide, while the third protein, a beta 2-microglobulin-associated glycoprotein of 43,000 is not yet defined by a monoclonal Ab. Other monoclonals described here include 3A1 and PVR-11, which are on subsets of peripheral T cell, and B1-192 and B1-499 which are on some activated T cells.

Unusual expression of human lymphocyte antigen class II in normal renal microvascular endothelium.

BACKGROUND: The human lymphocyte antigen (HLA) class II proteins (DR, DQ, and DP) and DM, a protein involved in loading antigenic peptide onto the class II molecules, have a coordinate regulation that facilitates antigen presentation to CD4+ T cells. CIITA is a specific transcription factor responsible for the coordinate regulation of these genes. DR expression in the kidney was described to be constitutive on renal microvascular endothelium in the early 1980s, but expression of other genes involved in class II antigen presentation (DQ, DP, DM, and CIITA) has not been characterized. METHODS: Expression of the HLA class II proteins, DM, and CIITA in normal human kidney cortex was evaluated by immunofluorescence microscopy, Northern blots, and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: The endothelium of glomerular and peritubular capillaries constitutively express DR, as indicated by colocalization of DR and CD31 antibodies. However, the endothelium of larger renal blood vessels is devoid of class II proteins. Capillaries that express DR do not have detectable DQ, DP, or DM by immunofluorescence. Northern blots identified DR, DP, and DM mRNAs but not DQ mRNA. CIITA was amplified by RT-PCR at a level that could account for the class II expressed by the microvascular endothelium. CONCLUSION: The renal microvascular endothelium constitutively expresses DR without the other class II proteins or DM. This discoordinate expression of HLA class II genes is unusual and may contribute to the kidney's ability to control CD4+ T-cell responses.

mRNA abundance, rather than differences in subunit assembly, determine differential expression of HLA-DR beta 1 and -DR beta 3 molecules.

The class II major histocompatibility molecules HLA-DR are formed by the association of a single DR alpha chain with two nonallelic DR beta chains. In DR3 cells one DR beta chain is severalfold more abundant than the other. We have studied the mechanism that controls the differential expression of these DR beta genes. We determined the amino-terminal sequences of the two expressed DR beta chains. Comparison of these sequences with the nucleotide sequences of the DR3B1 and DRB3a genes indicates that the abundant chain is the B1 gene product. Supporting this conclusion, an informative mutant, 9.4.3, was found to have lost the abundant beta chain and beta 1 mRNA. This mutant expresses normal cell surface levels of the DR beta 3 chain and exhibits no significant dosage compensation of its beta 3 chain. The unchanged level of DR beta 3 dimer on the cell surface suggests that free DR alpha chains are not the limiting factor in the surface expression of the beta 3 chain and, further, that the differential regulation of the surface expression of the two DR beta chains occurs at a step prior to DR assembly. Quantitation of DR beta mRNAs by locus-specific oligonucleotide probes showed that beta 1 mRNA is 4.5-fold more abundant than beta 3 mRNA, strongly indicating that the greater surface expression of DR beta 1 is a direct consequence of greater beta 1 mRNA abundance.

Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate.

The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody we found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, we studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalization of CD20 molecules; both phosphorylation and internalization of CD20 molecules were decreased with the protein kinase C inhibitor palmitoyl carnitine. Conditions that increase CD20 phosphorylation are shown also to increase surface mobility of the molecule, suggesting that CD20 protein internalization may be a critical early event for B-cell entry into the G1 phase of the cell cycle.

Mouse myeloma proteins with lambda 2 and lambda 3 light chains.

Serum and ascites fluid from mice bearing 260 different myeloma tumors were screened serologically for myeloma proteins having light chains like that of L315, the lambda 2 chain produced by myeloma MOPC-315. Four proteins, made by myelomas TEPC-952, CBPC-49, ABPC-72, and SAPC-15, were identified. Their light chains were essentially indistinguishable serologically from L315, and each of them also yielded the characteristic C-terminal amino acid (leucine) and C-terminal tryptic peptide of L315. However, amino acid sequences and other findings reported elsewhere have revealed that although the light chain of TEPC-952 is indeed a lambda 2 chain, the light chains of the others (CBPC-49, ABPC-72, and SAPC-15) represent another, slightly different type of light chain, which has been designated lambda 3.

Human T cell surface antigens bearing a structural relationship to HLA antigens.

Three cell surface antigens that are structurally related to the human major histocompatibility antigens (called HLA antigens) have been characterized from the leukemic T cell line MOLT-4. One antigen is a glycoprotein of Mr 49,000 recognized by two monoclonal antibodies. OKT6 and NA1/34, and is associated with a Mr 12,000 subunit that crossreacts serologically with beta 2-microglobulin but can be distinguished from it by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A second antigen, defined by the monoclonal antibody OKT10, is a Mr 46,000 protein associated with a small subunit distinct from beta 2-microglobulin. The OKT10 antigen is not restricted to T cells and is found on all T and B lymphoblastoid cell lines tested. The third protein is a beta 2-microglobulin-associated glycoprotein of Mr 43,000 that is serologically distinct from the OKT6 (NA1/34), OKT10, and HLA antigens. It is found on some, but not all, T cell lines but is absent from any other hematopoietic cell lines tested.

Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes.

Presentation of an exogenous protein antigen to helper (CD4+)T-lymphocytes by antigen presenting cells (APC) generally requires that the APCs degrade the native protein antigen into an immunogenic peptide, a process termed 'antigen processing', and that this peptide bind to a major histocompatibility complex (MHC) class II molecule. The complex of peptide and MHC molecule on the APC surface provides the stimulatory ligand for the alpha beta T cell receptor. The intracellular pathways and molecular mechanisms involved in the generation of the peptide-MHC complex are not well understood. Here, we describe several mutant APCs which are altered in their ability to present native exogenous protein antigens but effectively present immunogenic peptides derived from these proteins. The lesions in these mutants are not in the class II structural genes, but they affect the conformation of mature class II dimers.

Dual parameter flow cytometric analysis of DNA content, activation antigen expression, and T cell subset proliferation in the human mixed lymphocyte reaction.

This study provides direct correlation via dual parameter flow cytometry (simultaneous assessment of immunofluorescence and DNA content) between mixed lymphocyte reaction (MLR) responder cell entry into the S/G2/M phases of the cell cycle with the kinetics of expression of two activation-associated cell surface proteins, Tac (IL 2 receptor) and 4F2 (unknown metabolic function). A small population of activated cells was identifiable by expression of both Tac and 4F2 antigens before peak DNA synthesis in the MLR. This population of activation antigen-positive cells expanded linearly in size from days 3 to 7 of culture. Treatment of immature MLR cultures with anti-4F2 Mab and complement (C) before DNA synthesis (treatment on day 3, peak DNA synthesis on days 5 to 6) resulted in blunted proliferation and activation antigen expression when the same culture was analyzed after maturation on day 6, indicating that the activated population had been previously detected and removed by anti-4F2 Mab + C. The 4F2 antigen was expressed on a greater percentage of cells in the MLR at all times (days 3 to 9) than was Tac, was present on virtually all S/G2/M phase responder cells, and a large fraction of cells remained intensely 4F2+ subsequent to peak DNA synthesis. In contrast, after initially preceding responder cell entry into the S phase of the cell cycle, the kinetics of Tac antigen expression closely paralleled the kinetics of responder cell proliferation. A subpopulation of cycling responder cells was noted in all MLR cultures studied that expressed Tac antigen weakly or not at all. Cells within both T4 and T8 cell subsets proliferate with similar kinetics in response to alloantigen. The possibility that activation antigens can be utilized to study effector cell generation in the MLR and that this flow cytometric technique may be utilized to analyze the response to various alloantigens is discussed.

Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation.

This study describes the monoclonal antibody 5E9 and the cell surface antigen it defines. The hybridoma cell line T3-5E9 was derived from fusion of P3 X 63/Ag8 myeloma cells and spleen cells from BALB/c mice immunized with HSB-2 cells, a human T cell line. Although binding to only 1 to 5% of peripheral blood (PB) and spleen mononuclear cells, 5E9 antibody bound to 40 to 80% of Con A-, PHA-, or PWM-activated PB cells. Moreover, 5E9 antibody bound to variable numbers of Sezary, acute myelogenous leukemia, and ALL PB leukemia cells. 5E9 antibody bound to all hematopoietic and nonhematopoietic cell lines tested, to 11 +/- 1% of thymocytes, and 40% of nucleated bone marrow cells. Under reducing conditions, immunoprecipitation studies using 5E9 antibody demonstrated 5E9 antigen to be an 90,000 m.w. glycoprotein. Under nonreducing conditions, antigen 5E9 is a disulfide-linked dimer of approximately 190,000 daltons. Sequential precipitation experiments using antibody 5E9, alpha OD heteroantiserum (raised against T ALL cells), and monoclonal antibody OKT9 demonstrated that the 3 antibody preparations recognized the same 90,000 m.w. glycoprotein. Thus, antibody 5E9 defines an 90,000 m.w. human cell surface antigen that is absent on the majority of PB mononuclear cells and is expressed on rapidly dividing normal and malignant human cells. This monoclonal antibody should be a useful marker of human cell activation.