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Midwinter Conference of Immunologists
CLINICAL
IMMUNOLOGY
AND
Midwinter ESTHER Unirsersity
of California,
IMMUNOPATHOLOGY
18,
Conference
F. HAYS,
LEROY
133-144
(1981)
of Immunologists HOOD,
AND JAMES WATSON
Los Angeles. Laboratory of Nuclear Medicine and Radiation 900 Veteran Avenue, Los Angeles. California 90024 Received
May
Biology,
2 1. 1980
The Nineteenth Midwinter Conference of Immunologists was held January 26-29, 1980, at the Asilomar Conference Grounds, Pacific Grove, California, with Leroy Hood and James Watson as cochairmen. The subject of the conference was “Immunoglobulins: Genes to Receptors.” The conference included five half-day sessions by invited speakers and a poster session. The first session entitled “Antibody Genes” was opened with a discussion by Dr. Thomas Maniatis (California Institute of Technology) on the structure and linkage of mammalian globin genes. He used the model of the human globin gene family to probe the mechanisms of eucaryotic gene activation. There is a progression of globin gene products from hemopoietic cells of the blood islands in the yolk sac to the fetus and to the adult. Studies of the various types of Thalassemia syndromes hereditary anemias in which there is imbalance of synthesis of (Yand p globins) using the technics of molecular biology have given insight into the arrangement of globin genes on the chromosome and the mechanisms of gene activation. Philip Leder (National Institute of Child Health and Human Development) discussed the origin of antibody diversity. Formation of a complete immunoglobulin gene involves recombination between one of several hundred germline variable region genes and a joining site (J segment) encoded close to a constant region gene. This may generate antibody diversity somatically both by using different combinations of variable and J-region genes and by using alternative joining frames. The organization of mouse heavy chain genes was presented by Leroy Hood (California Institute of Technology). He pointed out that heavy chain genes have been isolated from sperm (undifferentiated) and myeloma (differentiated) libraries. Thus, the organization of VH, JH, and C, gene segments in DNA from both differentiated and undifferentiated cell sources can be compared. Two important observations were reported. First, an IgA producing myeloma cell (M603) has a heavy chain (Ygene that is composed of three contiguous gene segments-VH, JH, and C,, which are found in undifferentiated tissue as separate gene segments. This observation implies that two separate DNA rearrangements have occurred during the differentiation of the corresponding B-cell V-J joining and CH switching. Second, the variable region of the heavy chain of this myeloma immunoglobulin is encoded by three gene segments-v,, D, and JH, which are also separate gene segments in undifferentiated tissue. This finding supports the model of antibody diversity arising from the evolutionary variability of germline hypervariable133 0090-1229/81/010133-12$1.00/O Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
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region and framework genes and their combinatorial joining during somatic differentiation. Philip Early (California Institute of Technology) spoke about receptor and secreted IgM molecules as an example of eucaryotic RNA splicing. cDNAs derived from two species of F heavy chain in RNAs present in M104E myelomas have been cloned and sequenced. One of these cDNAs was shown to encode the secreted p heavy chain, and the other to encode a p chain with the properties of membrane-bound p chains. The two sequences differed only in the C terminal and 3’ untranslated positions of the mRNAs. The membrane p cDNA encodes a C terminus which is 41 amino acids long compared to the 20 amino acids of the secreted C terminus. The membrane p C terminus was shown to contain a block of 26 uncharged amino acids, which are probably inserted into the cell membrane. By a comparison of the cDNA sequences with sequences of the germline C, gene, Dr. Early has determined that both cDNAs are derived from the same C, gene. The mRNA for secreted p contains 187 nucleotides contiguous with the C,4 exon. By contrast, membrane CLmRNA utilizes an RNA splice site located between the C,4 and the secreted C terminus. It contains membrane C terminal sequences derived from two exons located about 2 kb 3’ to the C,4 exons. As a result of the different splicing patterns, membrane p mRNA is about 200 nucleotides longer than secreted p mRNA. This was confirmed by Northern blots of cytoplasmic RNA from Ml04E myeloma and W279 lymphoma cells. He concluded that the control of membrane versus secreted mRNA is exercised by the length of the transcript: if it includes the membrane C terminal exons. these are spliced to make membrane p mRNA, but if the transcript terminates or is cleared after the secreted C terminal sequences, the product is secreted p mRNA. Dr. Ursula Storb (University of Washington) presented data concerning the expression of K light chain genes. Previous findings in her laboratory had shown that in many myeloma cells more than one K gene is rearranged. In studies undertaken to determine whether all rearranged genes are expressed, she found that in the mouse myeloma NS-1, a subline of MOPC-21. three different rearranged K genes exist. In several sublines, in which each had lost one of these three genes, production of NS-1 K chains was correlated with the presence of one of the three K genes. Loss of this expressed gene eliminated the synthesis of NS-I K chains. loss of one of the other two rearranged genes did not. Dr. Storb reported further studies to determine if the unexpressed, rearranged K genes were in an “active” or “inactive” chromatin conformation. This was done by mild digestion of myeloma nuclei by DNase I. She found that rearranged K genes are DNase I sensitive. i.e., in an “active” chromatin conformation regardless of expression. and that germline K genes are DNase I insensitive. The rearranged-unexpressed genes therefore possess the signals for chromatin alterations which are apparently prevented from expression at a later step. Dr. Storb hypothesized that allelic exclusion generally operates by the functional rearrangement of one K gene; other rearrangements are relatively frequent, but they are generally nonfunctional, thus preventing the production of scrambled antibodies. It remains to be determined whether a specific mechanism exists to create functional rearrangements in pre-B cells.
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Dr. R. Maki (Base1 Institute for Immunology) discussed somatic rearrangement of immunoglobulin genes. To study rearrangements of DNA which take place to produce y2b immunoglobulin, a mouse myeloma which is a y2b subclass producer was used (MOPC 141). Two DNA rearrangements were found. In one, an embryonic V gene was rearranged at or near a short coding sequence called J. Sequence studies of the embryo-derived J’s revealed four heavy chain J’s. The 5’ flanking sequence of each J showed two short palindromes which were previously identified for light chain J’s. This finding suggested that the mechanism of V-J joining in heavy chains is similar, if not identical, to that in light chains. The other recombination event occurred between the 5’ flanking sequences of the C, gene and the 5’ flanking sequence of the CYZhgene. This event was referred to as the “Cp-CyZb switch recombination.” The sequence of this recombination point in both embryo and the rearranged y2b clone revealed that the mechanism for this recombination must be different from that of the V-J joining. The fate of the C,, gene in MOPC 141, a y2b producer, was analyzed by the Southern blot procedure using a C,, gene probe. The C,, gene had been previously mapped in the mouse embryo to the 5’ side of the C,,, gene. In MOPC 141 the C,, gene was not found. Therefore, Dr. Maki concluded that switch recombination in this myeloma involves deletion of the intervening DNA between C, and CY2,, genes. The next topic was that of RNA processing in immunoglobulin gene expression, presented by Randolph Wall (University of California, Los Angeles). He pointed out that it is now clear that immunoglobulin mRNAs. like almost all mRNAs studied to date, are generated from much larger nuclear RNA precursors by RNA splicing. Despite its essential role in higher eucaryotic gene expression, little is known of the signals and molecular events in the phenomenon of RNA splicing. Dr. Wall’s laboratory has approached this problem in several ways. One has involved comparative studies on immunoglobulin nuclear RNA splicing patterns in normal immunoglobulin-producing cells and in nonproducing variants. These variants may result from alterations in RNA splicing. He has found that the identical V+ C, intervening sequence is spliced out at different times in the processing pathways for two K mRNAs with the same J region but different V regions. Thus, the order of splicing steps appears to be determined by factors other than the specific intervening sequence involved. Dr. Wall reported that V-X splicing in heavy chain mRNA biogenesis occurs in several steps and is therefore more complex than in K mRNAs. This suggests that RNA splicing may generate new substrates for successive splicing events. A mechanism for nuclear RNA splicing was proposed. A specific small nuclear RNA, called V-l. recognized RNA splicing sites and base pairs with both ends of an intervening sequence so as to align them in exact register for cutting and splicing. He presented his recent findings suggesting the possibility that immunoglobulin gene expression may be regulated through post-transcriptional RNA processing and splicing. Dr. Suzanne Cory (Walter and Eliza Hall Institute) presented work done in collaboration with Dr. Jerry M. Adams on the arrangement and rearrangement of immunoglobulin genes. The organization of heavy chain genes in the germline were compared with those in immunoglobulin secreting cells. The numbers of VH and C, genes in the germline were investigated by analyzing fragments of DNA
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from 12- to 13-day mouse embryos and adult mouse liver using cloned DNA probes. The mouse genome was shown to contain a single copy of the C, gene, and probably also of the C, and the different C, genes. In contrast, two VH probes revealed distinct sets of VH genes, indicating that the germline contains multiple related V, genes. Multiplicity of VH genes was also demonstrated directly by cloning, which revealed that related V, genes were separated by a spacer sequence of about 14,000 base pairs. Dr. Cory stated that since rearrangement of immunoglobulin genes appears to be a prerequisite for expression, her laboratory sought evidence of changes in context of C, genes by comparing restriction fragments bearing C, genes in DNA of plasmacytomas with those of the embryo. C, sequences were found within rearranged DNA in an IgM secretor cell, as were C,, sequences in two IgA and several IgG secretor plasmacytomas. Unlike heavy chain expression, rearrangement was not confined to single alleles and often took two forms within one cell line. She next addressed the question of how a lymphocyte clone can switch from expression of one heavy chain to another while maintaining its V region specificity. It was pointed out that models for C,, switching include: variable splicing of a giant precursor RNA; movement of a single V,, gene; insertion of copies of a single V, gene near each C,, gene: and, in the deletion model, excision of DNA between the expressed VH and C, genes. The results from her laboratory strongly favor a deletion model. One plasmacytoma was found to be devoid of specific VH genes, and C, sequences were partially or totally deleted from seven IgA or IgG secreting plasmacytomas. The deletions were found to span various portions of the C,, locus in different lines. They were not confined to single C,-bearing chromosomes. The probable order of the C,, genes could be inferred. Deletions were shown to map near active C, genes, strongly suggesting that deletions constitute an integral part of the process which creates a functional V-C gene. The final topic of the antibody gene session was presented by Dr. Frederick R. Blattner (University of Wisconsin). He presented studies on the structure of the heavy chain locus of the mouse. His studies were conducted using cDNA plasmids constructed by reverse transcription of mRNAs of heavy chains of (Y,y2b, F, and 6. The mRNA was isolated from myelomas 5558, MPC 11, PC3741, and TPSE 1017. Each of the plasmid clones was confirmed by at least partial DNA sequence analyses. In each case, the cDNA included most of the constant region mRNA sequence, but only in the case of J558 was any part of the variable region included. In the JSSS-derived plasmid, DNA through the third hypervariable region including the J segment was present with the complete C region. This myeloma produces a dextran-binding immunoglobulin. Plasmids were used as probes to isolate clones from shotgun collections made with partially digested fragments of genomic DNA of mouse origin using the Charon 4A and the Charon 28 vector. In the case of y2b. both the plasmid (mRNA) and genomic DNA sequences were determined. In the case of the p genomic clones, several which also hybridized to the J segment DNA of the (Y cDNA clone were found. The hybridization was to a fragment of DNA about 8 kilobases in length located on the 5’ side of the p constant region. A section of about 1000 base pairs in that area was sequenced. A perfect match to the 5558 J segment was found as well as a second J region corresponding to the
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myeloma protein MOPC 315. Both of these DNA sequences were shown to have structural contents similar to the K and A light chain J regions. Some preliminary work on cloning 6 heavy chain genomic DNA was also presented. Professor Raymond D. Owen (California Institute of Technology) introduced the sixth Dan H. Campbell Memorial Lecture, given by Professor Linus Pauling (Linus Pauling Institute of Science and Medicine). Dr. Pauling presented an overview of the early work in immunochemistry at the California Institute of Technology to which he contributed so much. His talk provided a fitting historical background for the conference theme. The third session dealt with antibody diversity. The opening paper was presented by Dr. Barbara Birshstein of the Albert Einstein College of Medicine who gave a view of immunoglobulin gene structure and expression through the analyses of variant myeloma heavy chains. Her laboratory has isolated several variants of the MPC 11 mouse myeloma cell line which have been shown to synthesize altered immunoglobulin heavy chains. In contrast to the parental y2b heavy chain of 55,000 MW, she described some altered heavy chains of 50,000 or 40,000 MW lacking normal subclass-specific serological markers. Other variant chains were found to be the same size as the parent chain but had serological characteristics of a second subclass, y2a, indicative of the activation of previously silent genetic information. All of these variant proteins were found to be related to each other as well as to the parent immunoglobulin since they shared the parental idiotype, indicating the retention of at least part of the original variable region gene. Primary structural studies of a short heavy chain of 40,000 MW showed that it lacked the complete CH3 domain, and suggested that it arose by a frame shift followed by premature termination at an exon-intron boundary. Studies of the y2a variant proteins indicated that while some may contain complete y2a constant regions, others actually were y2b-y2a hybrid chains. In one variant, the junction between these two sequences was determined to lie within the CH2 domain and it was found to be comprised of 24 amino acids which, with one possible exception, seemed to be identical between y2b and y2a sequences. Dr. Birshstein’s data suggested that this junction may offer a site for recombinational events. She felt that analyses of these variants could allow dissection of physiologically important mechanisms, such as heavy chain class switches in normal lymphocytes. The arsenate system of A/J mice was discussed by Pila Estess (Southwestern Medical School, University of Texas). She used this as a model for the molecular dissection of an idiotypic determinant on the immunoglobulin molecule. Ms. Estess and Dr. J. Donald Capra have performed amino terminal amino acid sequence analyses on the heavy and light chains of induced monoclonal antibodies with specificity for the hapten p-azophenylarsonate. Four of eight of these antibodies have been found to react with conventional antisera to the previously described A/J anti-arsonate cross-reactive idiotype (CRI). Of the 16 chains analyzed, all but one contained sequence differences in their first framework segment (residues l-30) which distinguish them from heavy and light chain sequences found in anti-arsonate antibodies isolated from A/J serum or ascites fluid. The presence of such framework differences appeared to be independent of the presence of CRI. In spite of the framework substitutions, all four of the CRI-positive hybridoma
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antibodies were found to have V region frameworks that were very similar to each other and to the CR1 positive molecules found in A/J serum. Two of the four CRI-negative hybridoma antibodies were equally similar to the CRI-positive antibodies in their framework portions. However, the first hypervariable regions of the CRI-positive antiarsonate hybridomas were identical to those of the serum antibodies, while the CRI-negative hybridomas had at least one hypervariable region sequence that was different from CRI-positive molecules. Serological analyses with anti-idiotypic antisera generated against CRI-positive hybridoma products indicated that each monoclonal antibody may possess individual antigenic specificities different from the determinants detected with the conventional rabbit anti-CRI. It was pointed out that the consistent appearance of framework substitutions in what had been thought to be a homogeneous antibody population has important implications for our understanding of the generation of antibody diversity and for the precise chemical definition of an idiotype. The diversity of dextran antibodies was discussed by Mr. J. Schilling (California Institute of Technology). He reported that essentially complete variable region amino acid sequences have been obtained from 18 LY1.3 dextran-binding myeloma and hybridoma heavy chains. These sequences suggested to Mr. Schilling and his co-workers that the complete variable region gene was assembled from three discrete gene segments termed V, D. and J, which code for amino acids l-99, 100 and 101, 102- 117. respectively. Five V, eleven D, and five J sequences were found in the 18 dextran-binding proteins examined. No two of the eighteen variable region sequences were identical, suggesting that the combinatorial joining of three gene segments contributes significantly to antibody diversity. Each of these antibodies had similar, if not identical, A light chains. In a second study, myeloma proteins Ml04E and 5558 were used to define individual idiotypes (Id11 unique to each of the antibodies and a cross reactive idiotype (IdX) shared by the two proteins. The Id1 determinants correlated with residues encoded by the D segment, while the IdX determinant correlated with the presence of a carbohydrate attachment site and its flanking residues in the second hypervariable region of the heavy chains. The final paper in the antibody diversity session, presented by Dr. Martin Weigert (Institute for Cancer Research, Philadelphia), dealt with transcription and translocation of K chain genes. He pointed out that studies of nuclear RNA of mouse plasmacytomas secreting V,21 chains had demonstrated that the size of the K mRNA precursor depends on which of the four J, segments is used. This observation, he stated, is consistent with the view that the intervening sequence between C, and the J, is excised during RNA processing. These same studies also showed that about half of the plasmacytomas produced a nuclear RNA containing the C, sequence which was estimated to be about 9 kilobases. Dr. Weigert and his collaborators have now characterized these nuclear RNA components in more detail. This was done by using a cloned C, probe and V,21, 52, and J,-C, intervening sequence probes. The K mRNA precursors were directly identified using the V,21 probe. The large nuclear RNA, now estimated to be 8.4 kilobases, contained C,, J,. and C,-J, intervening sequences but not V,21 sequences. Hence, the 8.Ckilobase component is not a precursor of the K mRNA; instead it
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appears to be transcribed from the germline K allele of the expressed K locus. Dr. Weigert based this interpretation on the following correlation: All plasmacytomas in which the 8.4-kilobase RNA is observed have only one rearranged K locus; those which do not have this large RNA have rearranged both K loci, in which case, only one of the rearranged loci is transcribed. Dr. David Baltimore (Massachusetts Institute of Technology) began the session on lymphocyte differentiation by discussing his studies which analyze steps in the differentiation of B lymphocytes. He utilized several murine tumor cell lines and fetal liver cells to obtain B cells at various differentiation stages for study. The studies demonstrated that p heavy chain expression precedes light chain expression during B-lymphocyte ontogeny. Two forms of p mRNA were demonstrated: one encodes a C terminal segment appropriate for secretion of IgM and the other encodes a C terminal segment appropriate for membrane insertion of IgM. The proportions of these two messages vary in cells of different function so that B lymphocytes have a preponderance of the form appropriate for membrane binding and plasmacytomas have a preponderance of the form appropriate for secretion. Dr. Baltimore also reported his finding regarding the expression of K mRNA in A-secreting myelomas. All A myelomas, except MOPC 315, were found to have K mRNA. In two cases, the mRNA could be shown to encode fragments of K protein. He has interpreted these data to mean that cells sequentially switch on their light chain genes until a functional light chain is produced. At that time, the light chain combines with the preexisting p heavy chain in the cell and surface immunoglobulin appears. When surface immunoglobulin appears no new light chains are activated and thus any given cell clone will express only a single light chain. Dr. Marian Koshland (University of California, Berkeley), then reported her findings regarding activating J chain synthesis in B lymphocytes. Virgin B cells bearing surface immunoglobulin interact with antigen; the secreted p message appears and with appearance of secreted p there is a triggering of gene transcription for J chain synthesis. This J chain is required for assembly of the first antibody product of the differentiating B lymphocyte, namely pentomeric IgM. The ontogeny of the cellular expression of immunoglobulin genes was discussed by Max D. Cooper (University of Alabama). He reviewed the sites of induction of B cell differentiation in birds and mammals. Differences in immunoglobulin expression by the first recognizable cells of B lineage in birds and mammals were pointed out. Bursal pre-B cells in birds express IgM on their surface while mammalian pre-B cells from fetal liver express p chains in their cytoplasm but not on their surface. These pre-B cells lack characteristic B-cell surface components including Fc and C3 receptors and, in mice, Ia determinants. Large rapidly dividing pre-B cells give rise to progeny of a similar phenotype which replicate more slowly before expressing the cell surface IgM characteristic of B lymphocytes of the next stage of development. Murine and human pre-B cells which contain cytoplasmic p chains fail to reveal light chains suggesting, as did the findings of Dr. Baltimore, that the onset of p and light chain synthesis is asynchronous during ontogeny. Evidence for allelic exclusion at the pre-B-cell stage in rabbits was presented. The third topic in the lymphocyte differentiation session, factors controlling
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repertoire expression in B cells, was presented by Dr. Norman R. Klinman (Scripps Clinic and Research Foundation). Studies of the expressed antibody clonotype repertoire within individuals of inbred mouse strains have led to two important and surprising conclusions. First, as measured by any variety of approaches, the repertoire of individuals and the strain as a whole appears extremely large, encompassing more than IO7 unique specificities. Second, in spite of this enormous size, it appears that repertoire expression among genetically identical individuals is maintained with extraordinary fidelity, i.e., while many examples exist of identical expression among individuals of a strain, no evidence, as yet, exists that genetically identical individuals may differ in their expression of the repertoire. Studies from his laboratory have documented these observations. Dr. Klinman pointed out that findings at the molecular level as presented in earlier papers in the meeting indicated that the reproducible inheritance of a large repertoire could be accounted for by the presence of a large number of variable regions within the germline, as well as a complex set of mechanisms, such as recombination and insertion at the DNA level, nucleotide exchanges at DNA joining regions, splicing at the RNA level, and polypeptide chain shuffling. Genetic inheritance of repertoire was emphasized and Dr. Klinman felt that the greatest influence on repertoire expression subsequent to genetically determined events is the deletion of specificities either by tolerance or by immunoregulatory phenomena. His laboratory has defined an immunoregulatory phenomenon which is present in mice subsequent to immunization. It suppresses the stimulation of primary B cells which are specific for the immunizing antigen and which are syngeneic to the immunized individual at the heavy chain allotype locus. The existence of such a phenomenon could be assessed by relative stimulation of allotype syngeneic vs allotype allogeneic primary B cells. It was demonstrated that a similar immunoregulatory phenomenon accumulates in aged mice, presumably as the result of normal environmental antigenic stimulatory events. In order to determine if these naturally acquired immunoregulatory phenomena affect repertoire expression, the repertoire found in mature splenic B cells was compared to that observed in the immature B-cell population of adult bone marrow. Using the response to dextran, it was shown that specificities which normally occur in the early bone marrow B-cell population are not expressed in mature splenic B cells. In addition, B cells bearing these specificities could be stimulated only in the environment of allotype allogeneic individuals, since mature animals. syngeneic in allotype, suppressed stimulation of the early specificities. These findings were thought to imply that naturally acquired immunoregulatory phenomena may indeed affect repertoire expression. Thus, individuals inherit an enormous B-cell repertoire. but expression of this repertoire may be shaped significantly by deletion of specificities due to either tolerance induction or naturally accumulated immunoregulatory phenomena. The last presentation in this session, by Dr. Alfred Nisonoff (Brandeis University), dealt with “Genetic and Cellular Studies of a Strain Specific Idiotype.” A strain-specific idiotype is present on the anti-p-azophenylarsonate antibodies (Anti-Ar) in all A/J mice. Expression of the idiotype (cross-reacting idiotype. CRI) is linked to both the V, and the IgH loci. Investigations by Estess and Capra of
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hybridoma products (HPs) have shown that molecules bearing this idiotype exhibit microheterogeniety with respect to N-terminal V-region amino acid sequences. Microheterogeniety has also been demonstrated with respect to the fine specificity of idiotypic determinants. CR1 (+) HPs cause at least 55% displacement of labeled serum antibody from its anti-idiotypic antibody. In two fusions about 25% of hybridomas secreting anti-Ar antibodies were CR1 (+). In two pools of serum antibody over 80% of CR1 (+) antibodies were IgG,, which focuses at pZ 6.65 to 6.9. Individual HPs were found to possess private idiotypic determinants as well as the shared determinants characteristic of the CRI. The shared determinants are, in general, cross-reactive rather than identical, although some HPs are serologically very similar. Several HPs can react with almost the entire population of conventional anti-idiotypic antibodies, indicating that such antibodies are directed mainly to public rather than private determinants. In some of his recent studies of regulation, Dr. Nisonoff was able to show that CR1 could be selectively suppressed by inoculation of idiotype-conjugated thymocytes without any major suppression of the total anti-Ar population. Antigen and anti-idiotypic antibody were not required. Such suppressed mice produced idiotype specific T-suppressor cells which could adoptively transfer the suppressed state. Suppression by idiotype-conjugated thymocytes was demonstrated in several mouse strains. No genetic restriction on the source of thymocytes conjugated to the idiotype was observed. Pretreatment with antigen prevented suppression of idiotype by the conjugated cells suggesting that the secondary cells were resistant to idiotypic suppression. Dr. Nisonoff told of studies done in collaboration with Drs. Sy, Green, and Benacerraf which demonstrated that an Ar-specific T-cell-derived suppressor factor (TsF) in A/J mice carries the CRI. Inheritance of idiotypic determinants on TsF was found to be linked to the IgH locus, but not to the V, locus. The final topic of the meeting was that of antigen-binding receptors. The first speaker was Dr. Ellen Vitetta (University of Texas, Southwestern Medical School). Her topic was receptor-mediated triggering of B lymphocytes in the mouse. She stated that the vast majority of B lymphocytes in the spleen of adult mice coexpress IgM and IgD on their surface. In contrast, the majority of B cells in the spleens of neonatal mice bear only IgM. Experiments were performed to determine the Ig phenotype of B cells from adult and neonatal mice which respond to the thymus-dependent (TD) antigen, TNP-SRBC, and the thymus-independent (TI) antigens, TNP-BA and TNP-LPS. Normal adult spleen cells were treated with C’ and either anti-p or anti-6 to delete cells bearing IgM or IgD, respectively. Surviving cells were assessed for their capacity to respond to the TI and TD antigens either in vim or in vivo. The results indicated that the vast majority of the immunocompetent B cells from both adult and neonatal mice coexpress IgM and IgD. Further experiments were designed to determine the role of the two Ig receptors in triggering B lymphocytes. Earlier experiments utilizing isotypespecific antibodies to block in vitro responses to TD and TI antigens had suggested that cells that respond to TD antigens require both IgM and IgD for triggering whereas responses to some TI antigens require only IgM for triggering. Dr. Vitetta postulated that those TI antigens which can bypass the requirement for IgD re-
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ceptors could accomplish this by binding to mitogen receptors on B cells or, alternatively, TI antigens with a sufficiently high epitope could trigger cells in the absence of IgD by effectively crosslinking IgM receptors. The effect of epitope density of the antigen on the requirements for T-cell help and surface IgD on B cells was investigated using the antigen TNP-polyacrylamide beads (TNP-PAB). The PAB were not mitogenic for murine spleen cells and could be rendered either TD or partially TI by derivatizing the carrier with different numbers of TNP determinants. Dr. Vitetta’s results suggest that IgD and T cells are required for the B cell response when the epitope density of the antigen is low. In contrast, an increase in epitope density of the antigen is associated with a decreased requirement for both IgD and T cells. Based on these results, it was hypothesized that IgM receptors on B cells are functionally univalent and thus ineffectively crosslinked by paucivalent (low epitope density) antigens. IgD receptors, on the other hand, may be divalent and thus more readily crosslinked by paucivalent antigens. If, as other studies have suggested, crosslinking of ig receptors is a prerequisite for B-cell activation, multivalent antigens could activate B cells via IgM receptors, while IgD would be required to trigger cells with paucivalent antigens. Dr. Sandra Ewald (Montana State University), the next speaker, compared membrane-bound and secreted IgM molecules. She pointed out that F chains of membrane and secreted IgM have been compared structurally to determine the biochemical basis for their different localization. A mouse B-cell lymphoma (W279). characterized initially as an early B-cell tumor, was used as a source of membrane IgM. Secreted IgM with the same variable region was obtained from a hybridoma derived from fusion of W279 with an IgG-secreting myeloma. From the B-cell tumor three types of p chains were obtained. They differed in molecular weight and isoelectric point and/or in location (intracellular, membrane, or secreted). Only the secreted F chain from the hybridoma was studied. Structural studies were carried out by labeling cells biosynthetically with radioactive amino acids or mannose. Sodium borohydride to label galactose residues was used for extrinsic labeling. Cyanogen bromide fragments of each of the p forms labeled by the various means were separated by gel filtration and the profiles compared. The results showed that membrane p is missing the carboxy-terminal carbohydrate present on the other forms of immunoglobulin. Tunicamycin-treated W279 cells were found to produce two unglycosylated polypeptide chains differing in molecular weight. Dr. Ewald said that these data, as well as other corroborating evidence, suggested to her that the malignant B cells synthesize two forms of /-L chains, one of them destined for the membrane and the other possibly for secretion. A discussion of antigen-specific molecules from murine T lymphocytes and T-cell hybridomas was presented by Dr. Joel W. Goodman (University of California, San Francisco). Strain A/J mice immunized with azobenzenearsonate (ABA)-isologous IgG conjugates were found to make strong antigen-specific suppressor T-cell responses. The ABA-specific T cells were enriched by affinity purification and an average of 50% of such cells expressed the major idiotype (CRI) associated with anti-ABA specificity in A/J mice. The cellular proteins of
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affinity enriched ABA-specific T cells were biosynthetically radiolabeled and ABA-binding molecules from cell lysates were purified by affinity chromatography. SDS-PAGE analysis of T-cell-derived ABA-binding molecules revealed a major invariant component with an apparent MW of about 92,000 (~92). This protein was found to be susceptible to proteolytic degradation as its recovery from the cell lysates required the presence of protease inhibitors. Serological analyses of p92 failed to reveal classical immunoglobulin heavy or light chain determinants or Ia markers. Evidence was presented that the class of molecules represented by p92 possess suppressor regulatory activity, probably in conjunction with a noncovalently associated smaller polypeptide chain. Affinity-enriched ABA-specific suppressor T cells were hybridized with the AKR mouse tumor line, BW5147. Ten hybridomas were obtained which formed rosettes with ABA-SRBC. Rosette formation was inhibitable with soluble ABA protein conjugates. Culture supernatants from three lines were tested for modulation of an in &ro primary anti-ABA response and exerted a strong antigen-specific suppressor activity. Thus, hybridoma cell lines have been obtained by Dr. Goodman which express an ABAspecific receptor and secrete antigen-specific regulatory factors. They offer promise, he stated, as sources of sufficient material for chemical characterization of antigen-specific T-cell molecules. The last speaker for the session on antigen-binding receptors was Dr. Klaus Eichmann from the Institute for Immunology and Genetics, Heidelberg, West Germany. He presented a serologic and genetic analysis of the T-cell receptor. He stated that for an understanding of the antigen recognition system of T cells information must be accumulated in three areas: (1) the structure of the T-cell receptor; (2) the genes encoding the T-cell receptor, and (3) the diversity of the T-cell repertoire. Regarding the T-cell receptor, research in his laboratory has demonstrated that anti-idiotypic antibodies directed toward determinants on the variable region of the immunoglobulin heavy chains are reactive with T-cell receptors. He has also demonstrated in recent studies that framework determinants of the mouse immunoglobulin heavy chain are present on T-cell receptors. The demonstration of these determinants involved several assay systems including inhibition of antigen binding to T cells, induction of T helper cells, induction of T suppressor cells as well as inhibition of helper function in ~?itro. With respect to the second area, i.e., genes encoding for T-cell receptors, Dr. Eichmann reported a number of experimental systems in which he could demonstrate that the VHassociated idiotypic determinants of the T-cell receptor are encoded by genes linked to allotype. In addition, he showed that the V,-associated framework determinants are encoded by genes linked to allotypes. Taken together, these data suggested that VH genes encode the V, regions of the T-cell receptors. Additionally, he presented evidence that idiotypes on T cells reactive to alloantigens are specified by the H-2 complex. These data suggested that, in addition to VH, the T-cell receptor may contain additional structural elements, perhaps encoded by the H-2 complex. Regarding the third area of information, the diversity of the T-cell repertoire, Dr. Eichmann mentioned that previous studies in other laboratories have shown that alloreactive T cells are extremely frequent. He asked the question as to whether T cells reactive to conventional antigens are similarly
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frequent. Using a limiting dilution of purified cells with subsequent growth in Con A supernatant medium he was able to show that T helper cells specific for Streptococcus A bacteria were at least two orders of magnitude less frequent than are alloreactive T cells. This finding has given rise to the notion that T-cell receptors are in some way precommitted to recognize MHC-coded structures. An aiternative possibility considered by Dr. Eichmann was that the pronounced differences in frequency of T cells recognizing the two different sets of antigens may suggest that T cells use two independently distributed receptor systems, one for recognition of MHC coded antigens and the other for conventional antigens. ACKNOWLEDGMENT This Conference under Contract
was supported NOOO14-80-G-0019.
in part
by
the
Office
of Naval
Research.
Biochemistry
Program
IMMUNOLOGY
AND
Midwinter ESTHER Unirsersity
of California,
IMMUNOPATHOLOGY
18,
Conference
F. HAYS,
LEROY
133-144
(1981)
of Immunologists HOOD,
AND JAMES WATSON
Los Angeles. Laboratory of Nuclear Medicine and Radiation 900 Veteran Avenue, Los Angeles. California 90024 Received
May
Biology,
2 1. 1980
The Nineteenth Midwinter Conference of Immunologists was held January 26-29, 1980, at the Asilomar Conference Grounds, Pacific Grove, California, with Leroy Hood and James Watson as cochairmen. The subject of the conference was “Immunoglobulins: Genes to Receptors.” The conference included five half-day sessions by invited speakers and a poster session. The first session entitled “Antibody Genes” was opened with a discussion by Dr. Thomas Maniatis (California Institute of Technology) on the structure and linkage of mammalian globin genes. He used the model of the human globin gene family to probe the mechanisms of eucaryotic gene activation. There is a progression of globin gene products from hemopoietic cells of the blood islands in the yolk sac to the fetus and to the adult. Studies of the various types of Thalassemia syndromes hereditary anemias in which there is imbalance of synthesis of (Yand p globins) using the technics of molecular biology have given insight into the arrangement of globin genes on the chromosome and the mechanisms of gene activation. Philip Leder (National Institute of Child Health and Human Development) discussed the origin of antibody diversity. Formation of a complete immunoglobulin gene involves recombination between one of several hundred germline variable region genes and a joining site (J segment) encoded close to a constant region gene. This may generate antibody diversity somatically both by using different combinations of variable and J-region genes and by using alternative joining frames. The organization of mouse heavy chain genes was presented by Leroy Hood (California Institute of Technology). He pointed out that heavy chain genes have been isolated from sperm (undifferentiated) and myeloma (differentiated) libraries. Thus, the organization of VH, JH, and C, gene segments in DNA from both differentiated and undifferentiated cell sources can be compared. Two important observations were reported. First, an IgA producing myeloma cell (M603) has a heavy chain (Ygene that is composed of three contiguous gene segments-VH, JH, and C,, which are found in undifferentiated tissue as separate gene segments. This observation implies that two separate DNA rearrangements have occurred during the differentiation of the corresponding B-cell V-J joining and CH switching. Second, the variable region of the heavy chain of this myeloma immunoglobulin is encoded by three gene segments-v,, D, and JH, which are also separate gene segments in undifferentiated tissue. This finding supports the model of antibody diversity arising from the evolutionary variability of germline hypervariable133 0090-1229/81/010133-12$1.00/O Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
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region and framework genes and their combinatorial joining during somatic differentiation. Philip Early (California Institute of Technology) spoke about receptor and secreted IgM molecules as an example of eucaryotic RNA splicing. cDNAs derived from two species of F heavy chain in RNAs present in M104E myelomas have been cloned and sequenced. One of these cDNAs was shown to encode the secreted p heavy chain, and the other to encode a p chain with the properties of membrane-bound p chains. The two sequences differed only in the C terminal and 3’ untranslated positions of the mRNAs. The membrane p cDNA encodes a C terminus which is 41 amino acids long compared to the 20 amino acids of the secreted C terminus. The membrane p C terminus was shown to contain a block of 26 uncharged amino acids, which are probably inserted into the cell membrane. By a comparison of the cDNA sequences with sequences of the germline C, gene, Dr. Early has determined that both cDNAs are derived from the same C, gene. The mRNA for secreted p contains 187 nucleotides contiguous with the C,4 exon. By contrast, membrane CLmRNA utilizes an RNA splice site located between the C,4 and the secreted C terminus. It contains membrane C terminal sequences derived from two exons located about 2 kb 3’ to the C,4 exons. As a result of the different splicing patterns, membrane p mRNA is about 200 nucleotides longer than secreted p mRNA. This was confirmed by Northern blots of cytoplasmic RNA from Ml04E myeloma and W279 lymphoma cells. He concluded that the control of membrane versus secreted mRNA is exercised by the length of the transcript: if it includes the membrane C terminal exons. these are spliced to make membrane p mRNA, but if the transcript terminates or is cleared after the secreted C terminal sequences, the product is secreted p mRNA. Dr. Ursula Storb (University of Washington) presented data concerning the expression of K light chain genes. Previous findings in her laboratory had shown that in many myeloma cells more than one K gene is rearranged. In studies undertaken to determine whether all rearranged genes are expressed, she found that in the mouse myeloma NS-1, a subline of MOPC-21. three different rearranged K genes exist. In several sublines, in which each had lost one of these three genes, production of NS-1 K chains was correlated with the presence of one of the three K genes. Loss of this expressed gene eliminated the synthesis of NS-I K chains. loss of one of the other two rearranged genes did not. Dr. Storb reported further studies to determine if the unexpressed, rearranged K genes were in an “active” or “inactive” chromatin conformation. This was done by mild digestion of myeloma nuclei by DNase I. She found that rearranged K genes are DNase I sensitive. i.e., in an “active” chromatin conformation regardless of expression. and that germline K genes are DNase I insensitive. The rearranged-unexpressed genes therefore possess the signals for chromatin alterations which are apparently prevented from expression at a later step. Dr. Storb hypothesized that allelic exclusion generally operates by the functional rearrangement of one K gene; other rearrangements are relatively frequent, but they are generally nonfunctional, thus preventing the production of scrambled antibodies. It remains to be determined whether a specific mechanism exists to create functional rearrangements in pre-B cells.
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Dr. R. Maki (Base1 Institute for Immunology) discussed somatic rearrangement of immunoglobulin genes. To study rearrangements of DNA which take place to produce y2b immunoglobulin, a mouse myeloma which is a y2b subclass producer was used (MOPC 141). Two DNA rearrangements were found. In one, an embryonic V gene was rearranged at or near a short coding sequence called J. Sequence studies of the embryo-derived J’s revealed four heavy chain J’s. The 5’ flanking sequence of each J showed two short palindromes which were previously identified for light chain J’s. This finding suggested that the mechanism of V-J joining in heavy chains is similar, if not identical, to that in light chains. The other recombination event occurred between the 5’ flanking sequences of the C, gene and the 5’ flanking sequence of the CYZhgene. This event was referred to as the “Cp-CyZb switch recombination.” The sequence of this recombination point in both embryo and the rearranged y2b clone revealed that the mechanism for this recombination must be different from that of the V-J joining. The fate of the C,, gene in MOPC 141, a y2b producer, was analyzed by the Southern blot procedure using a C,, gene probe. The C,, gene had been previously mapped in the mouse embryo to the 5’ side of the C,,, gene. In MOPC 141 the C,, gene was not found. Therefore, Dr. Maki concluded that switch recombination in this myeloma involves deletion of the intervening DNA between C, and CY2,, genes. The next topic was that of RNA processing in immunoglobulin gene expression, presented by Randolph Wall (University of California, Los Angeles). He pointed out that it is now clear that immunoglobulin mRNAs. like almost all mRNAs studied to date, are generated from much larger nuclear RNA precursors by RNA splicing. Despite its essential role in higher eucaryotic gene expression, little is known of the signals and molecular events in the phenomenon of RNA splicing. Dr. Wall’s laboratory has approached this problem in several ways. One has involved comparative studies on immunoglobulin nuclear RNA splicing patterns in normal immunoglobulin-producing cells and in nonproducing variants. These variants may result from alterations in RNA splicing. He has found that the identical V+ C, intervening sequence is spliced out at different times in the processing pathways for two K mRNAs with the same J region but different V regions. Thus, the order of splicing steps appears to be determined by factors other than the specific intervening sequence involved. Dr. Wall reported that V-X splicing in heavy chain mRNA biogenesis occurs in several steps and is therefore more complex than in K mRNAs. This suggests that RNA splicing may generate new substrates for successive splicing events. A mechanism for nuclear RNA splicing was proposed. A specific small nuclear RNA, called V-l. recognized RNA splicing sites and base pairs with both ends of an intervening sequence so as to align them in exact register for cutting and splicing. He presented his recent findings suggesting the possibility that immunoglobulin gene expression may be regulated through post-transcriptional RNA processing and splicing. Dr. Suzanne Cory (Walter and Eliza Hall Institute) presented work done in collaboration with Dr. Jerry M. Adams on the arrangement and rearrangement of immunoglobulin genes. The organization of heavy chain genes in the germline were compared with those in immunoglobulin secreting cells. The numbers of VH and C, genes in the germline were investigated by analyzing fragments of DNA
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from 12- to 13-day mouse embryos and adult mouse liver using cloned DNA probes. The mouse genome was shown to contain a single copy of the C, gene, and probably also of the C, and the different C, genes. In contrast, two VH probes revealed distinct sets of VH genes, indicating that the germline contains multiple related V, genes. Multiplicity of VH genes was also demonstrated directly by cloning, which revealed that related V, genes were separated by a spacer sequence of about 14,000 base pairs. Dr. Cory stated that since rearrangement of immunoglobulin genes appears to be a prerequisite for expression, her laboratory sought evidence of changes in context of C, genes by comparing restriction fragments bearing C, genes in DNA of plasmacytomas with those of the embryo. C, sequences were found within rearranged DNA in an IgM secretor cell, as were C,, sequences in two IgA and several IgG secretor plasmacytomas. Unlike heavy chain expression, rearrangement was not confined to single alleles and often took two forms within one cell line. She next addressed the question of how a lymphocyte clone can switch from expression of one heavy chain to another while maintaining its V region specificity. It was pointed out that models for C,, switching include: variable splicing of a giant precursor RNA; movement of a single V,, gene; insertion of copies of a single V, gene near each C,, gene: and, in the deletion model, excision of DNA between the expressed VH and C, genes. The results from her laboratory strongly favor a deletion model. One plasmacytoma was found to be devoid of specific VH genes, and C, sequences were partially or totally deleted from seven IgA or IgG secreting plasmacytomas. The deletions were found to span various portions of the C,, locus in different lines. They were not confined to single C,-bearing chromosomes. The probable order of the C,, genes could be inferred. Deletions were shown to map near active C, genes, strongly suggesting that deletions constitute an integral part of the process which creates a functional V-C gene. The final topic of the antibody gene session was presented by Dr. Frederick R. Blattner (University of Wisconsin). He presented studies on the structure of the heavy chain locus of the mouse. His studies were conducted using cDNA plasmids constructed by reverse transcription of mRNAs of heavy chains of (Y,y2b, F, and 6. The mRNA was isolated from myelomas 5558, MPC 11, PC3741, and TPSE 1017. Each of the plasmid clones was confirmed by at least partial DNA sequence analyses. In each case, the cDNA included most of the constant region mRNA sequence, but only in the case of J558 was any part of the variable region included. In the JSSS-derived plasmid, DNA through the third hypervariable region including the J segment was present with the complete C region. This myeloma produces a dextran-binding immunoglobulin. Plasmids were used as probes to isolate clones from shotgun collections made with partially digested fragments of genomic DNA of mouse origin using the Charon 4A and the Charon 28 vector. In the case of y2b. both the plasmid (mRNA) and genomic DNA sequences were determined. In the case of the p genomic clones, several which also hybridized to the J segment DNA of the (Y cDNA clone were found. The hybridization was to a fragment of DNA about 8 kilobases in length located on the 5’ side of the p constant region. A section of about 1000 base pairs in that area was sequenced. A perfect match to the 5558 J segment was found as well as a second J region corresponding to the
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myeloma protein MOPC 315. Both of these DNA sequences were shown to have structural contents similar to the K and A light chain J regions. Some preliminary work on cloning 6 heavy chain genomic DNA was also presented. Professor Raymond D. Owen (California Institute of Technology) introduced the sixth Dan H. Campbell Memorial Lecture, given by Professor Linus Pauling (Linus Pauling Institute of Science and Medicine). Dr. Pauling presented an overview of the early work in immunochemistry at the California Institute of Technology to which he contributed so much. His talk provided a fitting historical background for the conference theme. The third session dealt with antibody diversity. The opening paper was presented by Dr. Barbara Birshstein of the Albert Einstein College of Medicine who gave a view of immunoglobulin gene structure and expression through the analyses of variant myeloma heavy chains. Her laboratory has isolated several variants of the MPC 11 mouse myeloma cell line which have been shown to synthesize altered immunoglobulin heavy chains. In contrast to the parental y2b heavy chain of 55,000 MW, she described some altered heavy chains of 50,000 or 40,000 MW lacking normal subclass-specific serological markers. Other variant chains were found to be the same size as the parent chain but had serological characteristics of a second subclass, y2a, indicative of the activation of previously silent genetic information. All of these variant proteins were found to be related to each other as well as to the parent immunoglobulin since they shared the parental idiotype, indicating the retention of at least part of the original variable region gene. Primary structural studies of a short heavy chain of 40,000 MW showed that it lacked the complete CH3 domain, and suggested that it arose by a frame shift followed by premature termination at an exon-intron boundary. Studies of the y2a variant proteins indicated that while some may contain complete y2a constant regions, others actually were y2b-y2a hybrid chains. In one variant, the junction between these two sequences was determined to lie within the CH2 domain and it was found to be comprised of 24 amino acids which, with one possible exception, seemed to be identical between y2b and y2a sequences. Dr. Birshstein’s data suggested that this junction may offer a site for recombinational events. She felt that analyses of these variants could allow dissection of physiologically important mechanisms, such as heavy chain class switches in normal lymphocytes. The arsenate system of A/J mice was discussed by Pila Estess (Southwestern Medical School, University of Texas). She used this as a model for the molecular dissection of an idiotypic determinant on the immunoglobulin molecule. Ms. Estess and Dr. J. Donald Capra have performed amino terminal amino acid sequence analyses on the heavy and light chains of induced monoclonal antibodies with specificity for the hapten p-azophenylarsonate. Four of eight of these antibodies have been found to react with conventional antisera to the previously described A/J anti-arsonate cross-reactive idiotype (CRI). Of the 16 chains analyzed, all but one contained sequence differences in their first framework segment (residues l-30) which distinguish them from heavy and light chain sequences found in anti-arsonate antibodies isolated from A/J serum or ascites fluid. The presence of such framework differences appeared to be independent of the presence of CRI. In spite of the framework substitutions, all four of the CRI-positive hybridoma
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antibodies were found to have V region frameworks that were very similar to each other and to the CR1 positive molecules found in A/J serum. Two of the four CRI-negative hybridoma antibodies were equally similar to the CRI-positive antibodies in their framework portions. However, the first hypervariable regions of the CRI-positive antiarsonate hybridomas were identical to those of the serum antibodies, while the CRI-negative hybridomas had at least one hypervariable region sequence that was different from CRI-positive molecules. Serological analyses with anti-idiotypic antisera generated against CRI-positive hybridoma products indicated that each monoclonal antibody may possess individual antigenic specificities different from the determinants detected with the conventional rabbit anti-CRI. It was pointed out that the consistent appearance of framework substitutions in what had been thought to be a homogeneous antibody population has important implications for our understanding of the generation of antibody diversity and for the precise chemical definition of an idiotype. The diversity of dextran antibodies was discussed by Mr. J. Schilling (California Institute of Technology). He reported that essentially complete variable region amino acid sequences have been obtained from 18 LY1.3 dextran-binding myeloma and hybridoma heavy chains. These sequences suggested to Mr. Schilling and his co-workers that the complete variable region gene was assembled from three discrete gene segments termed V, D. and J, which code for amino acids l-99, 100 and 101, 102- 117. respectively. Five V, eleven D, and five J sequences were found in the 18 dextran-binding proteins examined. No two of the eighteen variable region sequences were identical, suggesting that the combinatorial joining of three gene segments contributes significantly to antibody diversity. Each of these antibodies had similar, if not identical, A light chains. In a second study, myeloma proteins Ml04E and 5558 were used to define individual idiotypes (Id11 unique to each of the antibodies and a cross reactive idiotype (IdX) shared by the two proteins. The Id1 determinants correlated with residues encoded by the D segment, while the IdX determinant correlated with the presence of a carbohydrate attachment site and its flanking residues in the second hypervariable region of the heavy chains. The final paper in the antibody diversity session, presented by Dr. Martin Weigert (Institute for Cancer Research, Philadelphia), dealt with transcription and translocation of K chain genes. He pointed out that studies of nuclear RNA of mouse plasmacytomas secreting V,21 chains had demonstrated that the size of the K mRNA precursor depends on which of the four J, segments is used. This observation, he stated, is consistent with the view that the intervening sequence between C, and the J, is excised during RNA processing. These same studies also showed that about half of the plasmacytomas produced a nuclear RNA containing the C, sequence which was estimated to be about 9 kilobases. Dr. Weigert and his collaborators have now characterized these nuclear RNA components in more detail. This was done by using a cloned C, probe and V,21, 52, and J,-C, intervening sequence probes. The K mRNA precursors were directly identified using the V,21 probe. The large nuclear RNA, now estimated to be 8.4 kilobases, contained C,, J,. and C,-J, intervening sequences but not V,21 sequences. Hence, the 8.Ckilobase component is not a precursor of the K mRNA; instead it
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appears to be transcribed from the germline K allele of the expressed K locus. Dr. Weigert based this interpretation on the following correlation: All plasmacytomas in which the 8.4-kilobase RNA is observed have only one rearranged K locus; those which do not have this large RNA have rearranged both K loci, in which case, only one of the rearranged loci is transcribed. Dr. David Baltimore (Massachusetts Institute of Technology) began the session on lymphocyte differentiation by discussing his studies which analyze steps in the differentiation of B lymphocytes. He utilized several murine tumor cell lines and fetal liver cells to obtain B cells at various differentiation stages for study. The studies demonstrated that p heavy chain expression precedes light chain expression during B-lymphocyte ontogeny. Two forms of p mRNA were demonstrated: one encodes a C terminal segment appropriate for secretion of IgM and the other encodes a C terminal segment appropriate for membrane insertion of IgM. The proportions of these two messages vary in cells of different function so that B lymphocytes have a preponderance of the form appropriate for membrane binding and plasmacytomas have a preponderance of the form appropriate for secretion. Dr. Baltimore also reported his finding regarding the expression of K mRNA in A-secreting myelomas. All A myelomas, except MOPC 315, were found to have K mRNA. In two cases, the mRNA could be shown to encode fragments of K protein. He has interpreted these data to mean that cells sequentially switch on their light chain genes until a functional light chain is produced. At that time, the light chain combines with the preexisting p heavy chain in the cell and surface immunoglobulin appears. When surface immunoglobulin appears no new light chains are activated and thus any given cell clone will express only a single light chain. Dr. Marian Koshland (University of California, Berkeley), then reported her findings regarding activating J chain synthesis in B lymphocytes. Virgin B cells bearing surface immunoglobulin interact with antigen; the secreted p message appears and with appearance of secreted p there is a triggering of gene transcription for J chain synthesis. This J chain is required for assembly of the first antibody product of the differentiating B lymphocyte, namely pentomeric IgM. The ontogeny of the cellular expression of immunoglobulin genes was discussed by Max D. Cooper (University of Alabama). He reviewed the sites of induction of B cell differentiation in birds and mammals. Differences in immunoglobulin expression by the first recognizable cells of B lineage in birds and mammals were pointed out. Bursal pre-B cells in birds express IgM on their surface while mammalian pre-B cells from fetal liver express p chains in their cytoplasm but not on their surface. These pre-B cells lack characteristic B-cell surface components including Fc and C3 receptors and, in mice, Ia determinants. Large rapidly dividing pre-B cells give rise to progeny of a similar phenotype which replicate more slowly before expressing the cell surface IgM characteristic of B lymphocytes of the next stage of development. Murine and human pre-B cells which contain cytoplasmic p chains fail to reveal light chains suggesting, as did the findings of Dr. Baltimore, that the onset of p and light chain synthesis is asynchronous during ontogeny. Evidence for allelic exclusion at the pre-B-cell stage in rabbits was presented. The third topic in the lymphocyte differentiation session, factors controlling
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repertoire expression in B cells, was presented by Dr. Norman R. Klinman (Scripps Clinic and Research Foundation). Studies of the expressed antibody clonotype repertoire within individuals of inbred mouse strains have led to two important and surprising conclusions. First, as measured by any variety of approaches, the repertoire of individuals and the strain as a whole appears extremely large, encompassing more than IO7 unique specificities. Second, in spite of this enormous size, it appears that repertoire expression among genetically identical individuals is maintained with extraordinary fidelity, i.e., while many examples exist of identical expression among individuals of a strain, no evidence, as yet, exists that genetically identical individuals may differ in their expression of the repertoire. Studies from his laboratory have documented these observations. Dr. Klinman pointed out that findings at the molecular level as presented in earlier papers in the meeting indicated that the reproducible inheritance of a large repertoire could be accounted for by the presence of a large number of variable regions within the germline, as well as a complex set of mechanisms, such as recombination and insertion at the DNA level, nucleotide exchanges at DNA joining regions, splicing at the RNA level, and polypeptide chain shuffling. Genetic inheritance of repertoire was emphasized and Dr. Klinman felt that the greatest influence on repertoire expression subsequent to genetically determined events is the deletion of specificities either by tolerance or by immunoregulatory phenomena. His laboratory has defined an immunoregulatory phenomenon which is present in mice subsequent to immunization. It suppresses the stimulation of primary B cells which are specific for the immunizing antigen and which are syngeneic to the immunized individual at the heavy chain allotype locus. The existence of such a phenomenon could be assessed by relative stimulation of allotype syngeneic vs allotype allogeneic primary B cells. It was demonstrated that a similar immunoregulatory phenomenon accumulates in aged mice, presumably as the result of normal environmental antigenic stimulatory events. In order to determine if these naturally acquired immunoregulatory phenomena affect repertoire expression, the repertoire found in mature splenic B cells was compared to that observed in the immature B-cell population of adult bone marrow. Using the response to dextran, it was shown that specificities which normally occur in the early bone marrow B-cell population are not expressed in mature splenic B cells. In addition, B cells bearing these specificities could be stimulated only in the environment of allotype allogeneic individuals, since mature animals. syngeneic in allotype, suppressed stimulation of the early specificities. These findings were thought to imply that naturally acquired immunoregulatory phenomena may indeed affect repertoire expression. Thus, individuals inherit an enormous B-cell repertoire. but expression of this repertoire may be shaped significantly by deletion of specificities due to either tolerance induction or naturally accumulated immunoregulatory phenomena. The last presentation in this session, by Dr. Alfred Nisonoff (Brandeis University), dealt with “Genetic and Cellular Studies of a Strain Specific Idiotype.” A strain-specific idiotype is present on the anti-p-azophenylarsonate antibodies (Anti-Ar) in all A/J mice. Expression of the idiotype (cross-reacting idiotype. CRI) is linked to both the V, and the IgH loci. Investigations by Estess and Capra of
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hybridoma products (HPs) have shown that molecules bearing this idiotype exhibit microheterogeniety with respect to N-terminal V-region amino acid sequences. Microheterogeniety has also been demonstrated with respect to the fine specificity of idiotypic determinants. CR1 (+) HPs cause at least 55% displacement of labeled serum antibody from its anti-idiotypic antibody. In two fusions about 25% of hybridomas secreting anti-Ar antibodies were CR1 (+). In two pools of serum antibody over 80% of CR1 (+) antibodies were IgG,, which focuses at pZ 6.65 to 6.9. Individual HPs were found to possess private idiotypic determinants as well as the shared determinants characteristic of the CRI. The shared determinants are, in general, cross-reactive rather than identical, although some HPs are serologically very similar. Several HPs can react with almost the entire population of conventional anti-idiotypic antibodies, indicating that such antibodies are directed mainly to public rather than private determinants. In some of his recent studies of regulation, Dr. Nisonoff was able to show that CR1 could be selectively suppressed by inoculation of idiotype-conjugated thymocytes without any major suppression of the total anti-Ar population. Antigen and anti-idiotypic antibody were not required. Such suppressed mice produced idiotype specific T-suppressor cells which could adoptively transfer the suppressed state. Suppression by idiotype-conjugated thymocytes was demonstrated in several mouse strains. No genetic restriction on the source of thymocytes conjugated to the idiotype was observed. Pretreatment with antigen prevented suppression of idiotype by the conjugated cells suggesting that the secondary cells were resistant to idiotypic suppression. Dr. Nisonoff told of studies done in collaboration with Drs. Sy, Green, and Benacerraf which demonstrated that an Ar-specific T-cell-derived suppressor factor (TsF) in A/J mice carries the CRI. Inheritance of idiotypic determinants on TsF was found to be linked to the IgH locus, but not to the V, locus. The final topic of the meeting was that of antigen-binding receptors. The first speaker was Dr. Ellen Vitetta (University of Texas, Southwestern Medical School). Her topic was receptor-mediated triggering of B lymphocytes in the mouse. She stated that the vast majority of B lymphocytes in the spleen of adult mice coexpress IgM and IgD on their surface. In contrast, the majority of B cells in the spleens of neonatal mice bear only IgM. Experiments were performed to determine the Ig phenotype of B cells from adult and neonatal mice which respond to the thymus-dependent (TD) antigen, TNP-SRBC, and the thymus-independent (TI) antigens, TNP-BA and TNP-LPS. Normal adult spleen cells were treated with C’ and either anti-p or anti-6 to delete cells bearing IgM or IgD, respectively. Surviving cells were assessed for their capacity to respond to the TI and TD antigens either in vim or in vivo. The results indicated that the vast majority of the immunocompetent B cells from both adult and neonatal mice coexpress IgM and IgD. Further experiments were designed to determine the role of the two Ig receptors in triggering B lymphocytes. Earlier experiments utilizing isotypespecific antibodies to block in vitro responses to TD and TI antigens had suggested that cells that respond to TD antigens require both IgM and IgD for triggering whereas responses to some TI antigens require only IgM for triggering. Dr. Vitetta postulated that those TI antigens which can bypass the requirement for IgD re-
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ceptors could accomplish this by binding to mitogen receptors on B cells or, alternatively, TI antigens with a sufficiently high epitope could trigger cells in the absence of IgD by effectively crosslinking IgM receptors. The effect of epitope density of the antigen on the requirements for T-cell help and surface IgD on B cells was investigated using the antigen TNP-polyacrylamide beads (TNP-PAB). The PAB were not mitogenic for murine spleen cells and could be rendered either TD or partially TI by derivatizing the carrier with different numbers of TNP determinants. Dr. Vitetta’s results suggest that IgD and T cells are required for the B cell response when the epitope density of the antigen is low. In contrast, an increase in epitope density of the antigen is associated with a decreased requirement for both IgD and T cells. Based on these results, it was hypothesized that IgM receptors on B cells are functionally univalent and thus ineffectively crosslinked by paucivalent (low epitope density) antigens. IgD receptors, on the other hand, may be divalent and thus more readily crosslinked by paucivalent antigens. If, as other studies have suggested, crosslinking of ig receptors is a prerequisite for B-cell activation, multivalent antigens could activate B cells via IgM receptors, while IgD would be required to trigger cells with paucivalent antigens. Dr. Sandra Ewald (Montana State University), the next speaker, compared membrane-bound and secreted IgM molecules. She pointed out that F chains of membrane and secreted IgM have been compared structurally to determine the biochemical basis for their different localization. A mouse B-cell lymphoma (W279). characterized initially as an early B-cell tumor, was used as a source of membrane IgM. Secreted IgM with the same variable region was obtained from a hybridoma derived from fusion of W279 with an IgG-secreting myeloma. From the B-cell tumor three types of p chains were obtained. They differed in molecular weight and isoelectric point and/or in location (intracellular, membrane, or secreted). Only the secreted F chain from the hybridoma was studied. Structural studies were carried out by labeling cells biosynthetically with radioactive amino acids or mannose. Sodium borohydride to label galactose residues was used for extrinsic labeling. Cyanogen bromide fragments of each of the p forms labeled by the various means were separated by gel filtration and the profiles compared. The results showed that membrane p is missing the carboxy-terminal carbohydrate present on the other forms of immunoglobulin. Tunicamycin-treated W279 cells were found to produce two unglycosylated polypeptide chains differing in molecular weight. Dr. Ewald said that these data, as well as other corroborating evidence, suggested to her that the malignant B cells synthesize two forms of /-L chains, one of them destined for the membrane and the other possibly for secretion. A discussion of antigen-specific molecules from murine T lymphocytes and T-cell hybridomas was presented by Dr. Joel W. Goodman (University of California, San Francisco). Strain A/J mice immunized with azobenzenearsonate (ABA)-isologous IgG conjugates were found to make strong antigen-specific suppressor T-cell responses. The ABA-specific T cells were enriched by affinity purification and an average of 50% of such cells expressed the major idiotype (CRI) associated with anti-ABA specificity in A/J mice. The cellular proteins of
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affinity enriched ABA-specific T cells were biosynthetically radiolabeled and ABA-binding molecules from cell lysates were purified by affinity chromatography. SDS-PAGE analysis of T-cell-derived ABA-binding molecules revealed a major invariant component with an apparent MW of about 92,000 (~92). This protein was found to be susceptible to proteolytic degradation as its recovery from the cell lysates required the presence of protease inhibitors. Serological analyses of p92 failed to reveal classical immunoglobulin heavy or light chain determinants or Ia markers. Evidence was presented that the class of molecules represented by p92 possess suppressor regulatory activity, probably in conjunction with a noncovalently associated smaller polypeptide chain. Affinity-enriched ABA-specific suppressor T cells were hybridized with the AKR mouse tumor line, BW5147. Ten hybridomas were obtained which formed rosettes with ABA-SRBC. Rosette formation was inhibitable with soluble ABA protein conjugates. Culture supernatants from three lines were tested for modulation of an in &ro primary anti-ABA response and exerted a strong antigen-specific suppressor activity. Thus, hybridoma cell lines have been obtained by Dr. Goodman which express an ABAspecific receptor and secrete antigen-specific regulatory factors. They offer promise, he stated, as sources of sufficient material for chemical characterization of antigen-specific T-cell molecules. The last speaker for the session on antigen-binding receptors was Dr. Klaus Eichmann from the Institute for Immunology and Genetics, Heidelberg, West Germany. He presented a serologic and genetic analysis of the T-cell receptor. He stated that for an understanding of the antigen recognition system of T cells information must be accumulated in three areas: (1) the structure of the T-cell receptor; (2) the genes encoding the T-cell receptor, and (3) the diversity of the T-cell repertoire. Regarding the T-cell receptor, research in his laboratory has demonstrated that anti-idiotypic antibodies directed toward determinants on the variable region of the immunoglobulin heavy chains are reactive with T-cell receptors. He has also demonstrated in recent studies that framework determinants of the mouse immunoglobulin heavy chain are present on T-cell receptors. The demonstration of these determinants involved several assay systems including inhibition of antigen binding to T cells, induction of T helper cells, induction of T suppressor cells as well as inhibition of helper function in ~?itro. With respect to the second area, i.e., genes encoding for T-cell receptors, Dr. Eichmann reported a number of experimental systems in which he could demonstrate that the VHassociated idiotypic determinants of the T-cell receptor are encoded by genes linked to allotype. In addition, he showed that the V,-associated framework determinants are encoded by genes linked to allotypes. Taken together, these data suggested that VH genes encode the V, regions of the T-cell receptors. Additionally, he presented evidence that idiotypes on T cells reactive to alloantigens are specified by the H-2 complex. These data suggested that, in addition to VH, the T-cell receptor may contain additional structural elements, perhaps encoded by the H-2 complex. Regarding the third area of information, the diversity of the T-cell repertoire, Dr. Eichmann mentioned that previous studies in other laboratories have shown that alloreactive T cells are extremely frequent. He asked the question as to whether T cells reactive to conventional antigens are similarly
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frequent. Using a limiting dilution of purified cells with subsequent growth in Con A supernatant medium he was able to show that T helper cells specific for Streptococcus A bacteria were at least two orders of magnitude less frequent than are alloreactive T cells. This finding has given rise to the notion that T-cell receptors are in some way precommitted to recognize MHC-coded structures. An aiternative possibility considered by Dr. Eichmann was that the pronounced differences in frequency of T cells recognizing the two different sets of antigens may suggest that T cells use two independently distributed receptor systems, one for recognition of MHC coded antigens and the other for conventional antigens. ACKNOWLEDGMENT This Conference under Contract
was supported NOOO14-80-G-0019.
in part
by
the
Office
of Naval
Research.
Biochemistry
Program