Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells.

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells.

Inhibition of Epstein-Barr virus (EBV) release from the P3HR-1 Burkitt's lymphoma cell line by a monoclonal antibody against a 200,000 dalton EBV membrane antigen.

In raising murine hybridoma antibodies against Epstein-Barr virus (EBV)-induced membrane antigens (MA), we found one antibody that blocked the release of infectious EBV from cultured P3HR-1 cells. This monoclonal antibody (mAb) recognized a 200 kD, phosphonoacetic acid-sensitive (late) MA, and did not directly neutralize virus without complement. When this mAb was added to 33 degrees C-cultured, spontaneously EBV-producing P3HR-1 cells, the intracellular expression of viral capsid antigen and infectious virus was not inhibited, but the appearance of infectious virus in the culture medium was significantly reduced. The duration of this suppression was dependent upon the concentration of the mAb, an effect being observed to a 1:4 X 10(5) titer of the ascites mAb preparation. A more acute effect of suppression of EBV release was observed in a second model of 12-o-tetradecanoyl phorbol-13-acetate and n-butyrate induction of EBV in 37 degrees C-cultured P3HR-1 cells. Again, intracellular infectious virus production was not inhibited, but the level of infectious virus in the culture medium was significantly reduced as early as 1 and 2 d of culture with antibody. This effect was reversed within 31 h after replacement of mAb-containing medium with fresh medium. This description of antibody-mediated inhibition of EBV release might lead to the characterization of another form of immune defense for the control of EBV infections.

Inhibition of antibody-dependent cellular cytotoxicity and immunoglobulin synthesis by an antiserum prepared against a human B-cell Ia-like molecule.

Rabbit antisera to the human B-cell-specific antigen complex, p23,30, was used to define further the functional heterogeneity of isolated human lymphocyte subpopulations. Specific depletion of p23,30-bearing cells from Ig-negative cell populations and Ig-negative, E rosette-negative (Null) populations by either complement-mediated lysis or by physical separation on goat antirabbit Fab immunoabsorbent columns, eliminates the antibody-dependent cellular cytotoxic (ADCC) function. Furthermore, binding of anti-p23,30 serum to the effector cell surface inhibits ADCC but does not interfere with EA rosette formation. Apparently p23,30 represents a cell surface site which is distinct from the Fc receptor but which is important in the triggering of ADCC. In addition, depletion of p23,30-bearing cells from unfractionated cell populations, Ig-positive B-cell populations and Ig-negative, E rosette-negative (Null) populations eliminates the capacity of these populations to secrete immunoglobulin during subsequent culturing. Thus both the Ig-secreting cells and the ADCC effector cells within the Ig-negative, E rosette-negative (Null) population reside in the same population of cells which bears the p23,30 antigen.

Peripheral human T cells sensitized in mixed leukocyte culture synthesize and express Ia-like antigens.

We have studied the modulation of Ia-like antigens on the surface membrane of human T cells responding in a one-way mixed leukocyte culture. A heterologous antiserum, (anti-p23,30), which is specific to HLA-D-related antigens and which is unreactive with normal peripheral T cells or thymocytes, was found to bind significantly to all T cells transformed in mixed leukocyte culture (MLC) as determined by indirect immunofluorescence on a fluorescence-activated cell sorter 1. Furthermore, cytotoxic T cells responsible for cell-mediated lympholysis were shown to react with anti-p23,30, whereas their unactivated progenitors did not. Immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis of a radioactive 29,000 and 34,000 dalton complex from MLC-primed T cells labeled with [35S]methionine indicated that allosensitized T cells synthesized these HLA-D-related antigens.

Isolation and immunologic characterization of a human. B-lymphocyte-specific, cell surface antigen.

In addition to HL-A antigens, another cell surface protein complex has been obtained from membranes of the human B-lymphoblast cell line IM-1. This complex which was solubilized with papain, consisted of polypeptides of 23,000 and 30,000 daltons (p23, 30). Rabbit antisera to this material precipitated from [35S]methionine-labeled detergent-solubilized cells, three proteins of 39,000, 34,000, and 29,000 daltons. These antisera were specifically cytotoxic for B lymphocytes of peripheral blood, for B-lymphoblast cell lines, and for EAC rosette receptor-positive surface Ig-negative (Null) lymphocytes. The p23,30 complex was not present on T lymphocytes, EAC rosette receptor-negative Null lymphocytes, or platelets. In addition, the p23,30 complex from several cell lines inhibited alloantisera from multiparous Amish women which had been shown to recognize non-HL-A, B-lymphocyte antigens. Some other properties of the anti-p23,30 sera antisera were described.

Distribution of Lyb-4.1 and other membrane antigens on murine lymphoid tumors.

The distribution of membrane antigens on 6 DBA/2-derived tumors (L1210, L5178Y, P815, ABLS 11, ABLS 12, and ABLS 13) was studied by direct cytotoxicity and quantitative absorption assays. Lyb-4.1 antigen was found solely on the L1210 tumor. Iad antigens were absent from all tumors, and H-2Kd and H-2Dd antigens were present on all tumors. Immunoglobulin was adsorbed to the ascites tumors and lost after 3 days or more in tissue culture. These studies were performed to characterize the distribution of DBA/2 membrane antigens on DBA/2-derived tumors as a base line for functional and chemical studies with these tumors and with their solubilized proteins.

Epstein-Barr virus infections in hairy cell leukemia patients in the presence of complement-dependent neutralizing antibody.

Immune system status was characterized in patients with hairy cell leukemia (HCL) with respect to explaining their chronic or recurrent infections with Epstein-Barr virus. Measures of cellular immune responsiveness for a group of 11 HCL patients were, in general, decreased when expressed as the proportion of tested patients with values less than 2 S.D. below mean values for a group of 17 healthy adults: T-cell enumeration, seven of 13; mitogen responsiveness of phytohemagglutinin, 10 of 11; concanavalin A, 10 of 11; pokeweed mitogen, 10 of 11; B-cell responsiveness by anti-immunoglobulin immunobead stimulation, two of six; responsiveness to streptolysin O antigen, four of seven; mixed-lymphocyte reaction, six of seven; natural killer cell activity, six of eight. Specific immunity to Epstein-Barr virus was measured by complement-independent, antibody-mediated virus neutralization (mean index for HCL patients being 56% of control value) and complement-dependent virus neutralization (98% of control value). We concluded that, in spite of depressed levels of immune responses measured with general, cellular assays, functional levels of complement-dependent virus-neutralizing antibody were present in these HCL patients.

Heterogeneity in the membrane proteins of human lymphoid cell lines as seen in sodium dodecyl sulfate-polyacrylamide electrophoresis slab gels.

The proteins of [35S]methionine-labeled membranes of six human lymphoid cell lines were examined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gradient slab gels in order to identify molecular differences among these tumors. The lymphoid cells were internally labeled with [35S]methionine, their membranes were isolated, and the reduced and alkylated membrane proteins were treated electrophoretically in sodium dodecyl sulfate-polyacrylamide gradient slab gels. The gel patterns of over 100 membrane proteins per cell were highly complex but reproducible and, in that sense, constituted fingerprints of the individual tumors. Several proteins occurred uniquely on one or a few tumors. Some protein bands were identified to be serologically recognized membrane antigens by electrophoresis of immunopurified antigen in parallel to membrane samples. p44,12, a complex of proteins with molecular weights of 44,000 and 12,000 (HLA-A and -B antigens and beta2-microglobulin), and p29,34, (HLA-D antigen) were identified in this manner. High-resolution sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis can be used to catalog and describe lymphocyte membrane proteins and perhaps to identify subsets of lymphoid cancers.

Assessment of homology with the helical mimicry algorithm.

Homologies based on structural motifs characterize conserved structures and mechanisms of maintaining function. An algorithm was developed to quantitate homology among segments of two proteins based upon structural characteristics of an amphipathic alpha-helix. This helical mimicry algorithm scored homology among sequences of two proteins in terms of: (i) presence of Leu, Ile, Val, Phe, or Met in a longitudinal, hydrophobic strip-of-helix at positions n, n + 4, n + 7, n + 11, etc. in the primary sequence, (ii) identity or chemical similarity of amino acids at intervening positions and (iii) exchanges of amino acids from positions n to n - 1, n + 3, n + 4, n + 1, n - 3, n - 4 around n (on the surface of a putative helix). While such exchanges of amino acids on the surfaces of homologous helices may conserve function, they did not maintain specific interactions of those residues with apposing groups.

Hyperexpressed hairy leukemic cell Ii might bind to the antigen-presenting site of class II MHC molecules.

The p35 protein which is hyperexpressed on hairy leukemic cells was determined to be Ii, the electrophoretically invariant glycoprotein that is associated with class II major histocompatibility complex (Ia) antigens from the time of their synthesis. The principal function of class II MHC antigens is to present to T cell receptors those digested foreign antigenic peptides that probably fold as amphipathic alpha-helices and adsorb to a hydrophobic surface (desetope) on Ia. By a novel strip-of-helix hydrophobicity algorithm we found that the sequence Leu-142 to His-170 in Ii formed a five-cycle, amphipathic, alpha-helix, the highest scoring one among a series of proteins commonly used as experimental antigens. This finding led to the hypothesis that this sequence in Ii bound to the antigen-binding site (desetope) of Ia until release and self-aggregation in the endosome in order that digested foreign peptides could then bind to Ia. Abundant expression of Ii in leukemic cells might be associated with an altered capacity of those cells to present foreign or leukemic antigens to the host's immune system.

Binding of radioiodinated influenza virus peptides to class I MHC molecules and to other cellular proteins as analyzed by gel filtration and photoaffinity labeling.

In order to determine how T cell-presented peptides associate with the antigen binding sites (desetopes) of class I major histocompatibility complex (MHC) molecules and how they might be scavenged from an endogenous processing pathway for transfer to those molecules, we characterized the binding of two synthetic peptides restricted by HLA-B37 or HLA-A2 to class I MHC molecules and to cellular proteins of histotyped cell lines, by gel filtration and photo-affinity labeling techniques. In gel filtration binding studies, each peptide associated with immunopurified class I MHC molecules from cells with its restricting, histotype, but little was bound to class I MHC molecules from cells without the restricting histotype and none was bound to bovine serum albumin. After crosslinkage of a radioiodinated photoreactive derivative of influenza virus nucleoprotein peptide NP(336-355Y) and immunoprecipitations with antibodies to class I MHC molecules, that peptide was found to bind to immunopurified class I MHC molecules from HLA-B37+ but not HLA-B37- cells. Binding of the [125I]NP peptide increased from 6 to 12 hr of incubation and was competed by unlabeled, NP peptide but not by HLA-A2-restricted, influenza virus matrix MA(57-73). The principal microsomal membrane proteins binding [125I]NP were about 65, 45 and 33 kD.

Characterization of the invariant chain C-terminus (Glu183-Glu193) epitope which is obscured in processed Ii, MHC alpha,beta trimers.

The E1 serum was developed against invariant chain peptide Ii (183-193) in order to study the function of the Ii protein which associates with class II MHC alpha,beta chains from time of synthesis until cleavage and release, possibly regulating the binding of antigenic peptides. Subpopulations of Ii, Ii(VIC) and Ii(E1), respectively, were demonstrated by sequential immunodepletions and immunoprecipitations with: (1) VIC-Y1 monoclonal antibody to an N-terminal epitope of Ii, and (2) E1 rabbit antiserum to Ii(183-193). In 3 hr radiolabeled cells, VIC-Y1 recognized Ii, Ii and N- and O-linked glycosylation (IpN, IpO), p41 and co-precipitated class II alpha,beta chains, while E1 recognized Ii, IpN and immature Ii-alpha complex. In 15 min radiolabeled cells, each antibody recognized similar, immature Ii forms without alpha,beta. Urea denaturation of Ii(VIC) rendered the main Ii species but not IpO immunoprecipitable with E1. E1 recognized O-glycanase-treated Ii (VIC). We conclude that the Ii(183-193) epitope was obscured by interactions of Ii with class II alpha,beta chains and by the O-linked glycosylation of Thr187, which may in part regulate association of Ii to class II alpha and beta chains.

Effects of brefeldin A on cleavage of invariant chain to p21 and p10 and the appearance of Ii-freed class II MHC dimers.

Intracellular cleavage of class II MHC-associated Ii to p21 and p10 and the appearance of Ii-freed alpha, beta dimers were concurrent events lasting from 1 to 6 hr after synthesis of alpha, beta, Ii trimers, possibly related to charging of foreign peptides to the class II MHC antigen-binding site. Sequential immunoprecipitations of pulse-chase radiolabeled cells were made four times with anti-Ii monoclonal antibody to remove Ii and alpha, beta, Ii trimers and then with anti-class II antibody to detect the time-dependent appearance of Ii-freed alpha, beta dimers. The cleavage of Ii to p21 and p10 was revealed in leupeptin-treated cells. Cell treatment with Brefeldin A (BFA) was associated with a decrease in Ii-freed alpha, beta dimers, with inhibition of leupeptin-revealed cleavage of Ii to p21 and p10, and with persistence of endoglycosidase H susceptibility of Ii and class II alpha, beta chains. We conclude that in untreated cells, cleavage and release of Ii from class II MHC alpha and beta chains occur after those complexes traverse a BFA-sensitive step in the Golgi apparatus.

More efficient peptide binding to MHC class II molecules during cathepsin B digestion of Ii than after Ii release.

The binding of a T cell-presented peptide to MHC class II alpha,beta chains occurs as a concurrent process with the release of the associated invariant chain (Ii) by cathepsin B. Ii was digested by cathepsin B from solubilized, MHC class II alpha,beta,Ii complexes in the presence of N-hydroxysuccinimidyl-4-azidobenzoate-conjugated, 125I-labeled, influenza virus matrix (18-29) peptide. The peptide was crosslinked where it became bound. This HLA-DR1-restricted peptide bound about three times more efficiently to class II alpha,beta chains of DR1-positive B cells when present during cathepsin B digestion of Ii than when added afterward, also at pH 5.0. Binding was competed by similarly DR-restricted peptides. Cathepsin D cleaved Ii but did not enhance peptide binding. However, a trace level of cathepsin D, added to the assay for peptide binding in the presence of cathepsin B, further enhanced peptide binding about three times. These experiments support an hypothesis for the staged release of Ii fragments by cathepsin D and cathepsin B, catalyzing at one point the insertion of a peptide into the antigen binding site formed by class II alpha and beta chains.

Selection of class I MHC-restricted peptides with the strip-of-helix hydrophobicity algorithm.

A strip-of-helix hydrophobicity algorithm to predict class II MHC-restricted peptides, on the basis of their structural similarity to an amphipathic, alpha-helix in Ii, also predicted peptides which were presented to cytotoxic T-cells by class I MHC molecules. This algorithm ranked peptides according to mean Kyte-Doolittle hydrophobicity values of amino acids at positions n, n + 4, n + 7, n + 11, n + 14 and n + 18 in a sequence which when coiled as a putative alpha-helix, had the indicated residues in an axial strip along one side of the helix. Sequences selected for highly scoring, hydrophobic strips were required to have at least 1 of the 4 adjacent strips scoring more negatively than -1 in the strip-of-helix hydrophobicity index and the entire sequence could contain no prolines. This algorithm predicted the class I MHC-restricted, T-cell-presented peptides in sequences of 4 proteins from which some class I MHC-restricted, T-cell-presented sequences had been experimentally determined. Since both class I and class II MHC-restricted peptides could be identified with this algorithm, one can propose that: (1) foreign peptide-binding sites (desetopes) of the class I and class II MHC molecules are structurally similar; and (2) any one T-cell-presented peptide can be presented by some specific allele of both a class I and a class II MHC antigen.

Recognition of disparate HA and NS1 peptides by an H-2Kd-restricted, influenza specific CTL clone.

CTLs (CD8+) are known to recognize exogenous peptide in the context of class I MHC molecules. We observed that an influenza subtype H1 and H2 cross-reactive CTL clone B7, which was stimulated by a fusion protein containing a portion of HA2 subunit of A/PR/8 virus HA, recognized a synthetic peptide (residues 515-526) of the HA2 subunit of A/PR/8 virus strain. This CTL clone also recognized a structurally disparate NS1 peptide 50-68 of the same A/PR/8 virus. We examined the recognition of the NS1 peptide 50-68 and the HA peptide 515-526 by the subcloned CTL clone, B7-B7. Cold target inhibition experiments showed that the recognition of the HA peptide by the CTL clone B7-B7 could be competed by NS1 peptide-treated target cells and vice versa. The recognition of both NS1 peptide and HA peptide by the CTL clone B7-B7 was restricted by the same allele, H2Kd. In addition, this NS1 peptide requires approximately a 600-fold higher concn for optimal CTL recognition than did the HA peptide. We conclude that the TCR on clone B7-B7 recognizes the HA peptide or the NS1 peptide as comparable complexes with the same class I MHC molecules, although there is no obvious homology in the primary sequences of HA 515-526 and NS1 50-68 peptides. CTLs elicited with certain antigens appear to recognize distinctively different antigens complexed to the same presenting MHC molecule.

Cathepsin B cleavage and release of invariant chain from MHC class II molecules follow a staged pattern.

A staged pattern of cathepsin B cleavage of MHC class II alpha, beta-bound invariant (Ii) chain and release of fragments was defined. Charge-loss mutations in the Ii chain were created in three clusters of cathepsin B putative cleavage sites R78K80K83K86, K137K143, and R151K154. Products of HLA-DR1 alpha, beta and wild type (WT) or mutant Ii genes, co-transfected into COS1 cells, were cleaved by cathepsin B and immunoprecipitated by antibodies either to MHC class II chains or to different Ii epitopes. In WT Ii, cathepsin B digestion generated two forms of p21 Ii fragments: a p21 recognized by anti-C-terminus antibodies and a p21 recognized by an antibody to a determinant near the N-terminus. C-terminal p21 was released from MHC class II alpha, beta chains upon its formation while N-terminal p21 remained associated with MHC class II alpha, beta chains. Mutations at K137K143 inhibited the generation of N-terminal p21 by cathepsin B. Mutation at R78K80K83K86 led to an accumulation of MHC class II-bound N-terminal p21 without the appearance of MHC class II-bound p14, p10, and p6 fragments after cathepsin B digestion. These results indicate that cathepsin B cleaves wild type Ii first about K137K143 to produce a MHC class II-associated N-terminal p21, which is then cleaved about R78K80K83K86 to generate p14, p10 and finally p6 which still associates with MHC class II alpha, beta chains. This pattern of staged cleavage and release of Ii might be related to a concerted mechanism regulating the binding of antigenic peptides to MHC class II molecules.

Hydrophobic strip-of-helix algorithm for selection of T cell-presented peptides.

In extension of the hypothesis that an amphipathic alpha helix of Ii (Phe146-Val164) bound to the foreign antigen-presenting site (desetope) of class II MHC molecules through hydrophobic amino acid residues (Phe146, Leu150, Leu153, Met157, Ile160, Val164) which were present in an axial strip along one side of the Ii helix, we developed an algorithm to search for T cell-presented peptides showing a similar hydrophobic strip-of-helix. Such peptides might bind to the class II MHC molecule site which was complementary to the Ii hydrophobic strip-of-helix. The strip-of-helix hydrophobicity index was the mean hydrophobicity (from Kyte-Doolittle values) of sets of amino acids in axial strips down sides of helices for 3-6 turns, at positions, n, n + 4, N + 7, n + 11, n + 14, and n + 18. Peptides correlating well with T cell responsiveness had: (1) 12-19 amino acids (3-5 cycles or 4-6 turns of an alpha helix), (2) a strip with highly hydrophobic residues, (3) adjacent, moderately hydrophilic strips, and (4) no prolines. The degree of hydrophilicity of the remainder of a putative antigenic helix above a threshold value did not count in this index. That is, the magnitude of amphipathicity was not judged to be the principal selecting factor for T cell-presented peptides. This simple algorithm to quantitate strip-of-helix hydrophobicity in a putative amphipathic alpha helix, allowing otherwise generally hydrophilic residues, predicted 10 of 12 T cell-presented peptides in seven well-studied proteins. The derivation and application of this algorithm were analyzed.

Comparison of three related methods to select T cell-presented sequences of protein antigens.

A comparison of three methods to predict T cell-presented sequences within antigenic proteins led to the view that recurrent hydrophobic residues might nucleate excised peptides as alpha-helices against hydrophobic surfaces. Such helices could be protease-protected structures on their way to desetope binding. The compared methods were: the amphipathicity algorithm of DeLisi and Berzofsky [Proc. natn. Acad. Sci. U.S.A. 82, 7048-7052. (1985)] as modified by Margalit et al. [J. Immun. 138, 2213-2229. (1987)] the strip of-helix hydrophobicity algorithm (SOHHA) of Stille et al. [Molec. Immun. 24, 1021-1027. (1987)] and the motifs algorithm of Rothbard and Taylor [EMBO J. 7, 93-100. (1988)]. Correct prediction was defined at two levels of stringency: (1) the predicted sequence overlapped the experimentally reported sequence when the ratio of the intersection of both to the union of both greater than or equal to 0.5 or (2) the sequences touched when there was a non-empty intersection of both sequences. We determined the sensitivity (correct predictions/number of reported T cell-presented sequences) and efficiency (correct predictions/number of predictions) at each level of stringency. In terms of overlap, the SOHHA was more sensitive (0.43) than the amphipathicity (0.29) (not significant) and motifs (0.0, 0.0) (p less than 0.05) predictions and more efficient (0.35) than the amphipathicity (0.14) and motifs (0.0, 0.0) predictions. At the less stringent criterion touching, the amphipathicity method (0.71) was as sensitive as motif Rothbard-4 (0.79) and more sensitive than SOHHA (0.57) and motif Rothbard-5 (0.43). At that criterion, the SOHHA was more efficient (0.47) than the amphipathicity (0.36) and motifs (0.25, 0.40) methods. We hypothesize that the comparability of these approaches reflected the common, predominant influence of recurrent hydrophobicity in their predictions.

Membrane and culture fluid proteins of nine human T lymphoblastoid cell lines.

Proteins form the membranes and culture media of nine, human T lymphoblastoid cell lines and of the myeloid line K562 were analyzed after 35S-methionine metabolic labeling, by electrophoresis in SDS polyacrylamide gradient gels. A monotonous consistency was present among these cell lines in their synthesis of membrane proteins, with only few variations in the expression of most proteins. In contrast, the synthesis of culture supernatant proteins was found to differ substantially among the cell lines. Proteins of 35,000 and 36,000 daltons were most prominently synthesized by cell line 1301. Proteins of 50,000 daltons were abundantly synthesized by SKW 3 and 8402. Proteins of 65,000 daltons were best synthesized by Jurgat, 1301 and CEM. Few of the membrane proteins which were abundantly synthesized by mitogen-activated normal T lymphocytes were expressed on the cultured cell lines. The view was proposed that cultured T lymphoblastoid malignancies do do not represent static vignettes of stages of differentiation of normal T lymphocytes.