New insights into signalling-pathway alterations in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and young adults. Several recent studies have shed new light on the alterations in signalling pathways and the downstream effects of these pathway alterations in RMS. Many of these effects converge on the fibroblast growth factor and insulin-like growth-factor pathways. These new findings improve the current understanding of RMS, thus offering novel potential therapeutic targets and strategies that may improve the outcome for patients with RMS.

CHEMICAL CHARACTERIZATION OF GLYCOPEPTIDES FROM HUMAN gammaM-GLOBULINS.

A survey of human pathological macroglobulins revealed that gammaM can be divided into at least two groups on the basis of carbohydrate composition. Differences between the two groups exist in the total percentage of carbohydrate (10.69 +/- 1.49% for group 1, 7.71 +/- 0.65% for group II) which is attributable to variation in hexose content. Glycopeptides from macroglobulins of each group were purified from pronase digests and characterized chemically. Macroglobulins from each group contain three types of oligosaccharides. Glycopeptide I for each group consisted of mannose, galactose, and NAG with a ratio of 3:2:1 for group I and a ratio of 2:1:2 for group II. Glycopeptide II consisted of mannose, galactose, and NAG (9:1:2) for group I, and mannose, fucose, galactose, and NAG (2:1:3:2) for group II. Glycopeptide III in both groups consisted of mannose, fucose, galactose, NAG, and sialic acid with a ratio of 6:2.5:2.5:5.5:2 for group I and a ratio of 5:1:1:6:1 for group II. Molecular weight estimations by gel filtration indicates that there are 10 glycopeptides I and II and 20 units of glycopeptide III per molecule of gammaM.

Clonal nature of the immune response to phosphorylcholine (PC). V. Cross-idiotypic specificity among heavy chains of murine anti-PC antibodies and PC-binding myeloma proteins.

Seven mouse myeloma proteins with specificity for phosphorylcholine (PC) were found to share a common antigenic determinant. This group of proteins contained members which differed in genetic origin, heavy chain class, kappa-chain subgroup, individual antigenic determinants and specificity for choline analogues. The cross-idiotypic determinant, VH-PC, was antigenically similar in each of the proteins and was associated with the variable portion of the heavy chain in the region of the antibody combining site. Further studies showed that an indistinguishable determinant was present on IgM anti-PC antibodies isolated from all strains of mice tested regardless of histocompatibility or heavy chain allotype. In view of the finding that this cross-idiotypic determinant was not found on antibodies or myeloma proteins which lacked specificity for PC, the data strongly suggest that a particular heavy chain variable region has been preserved in all mouse antibodies with specificity for PC.

Clonal nature of the immune response to phosphorylcholine. IV. Idiotypic uniformity of binding site-associated antigenic determinants among mouse antiphosphorylcholine antibodies.

A new idiotypic determinant(s) on mouse anti-PC antibodies is described. Antibodies to the determinant(s) were raised in rabbits by immunization with HOPC 8, a PC-binding myeloma protein, and were isolated from HOPC 8 immunoadsorbent by elution with PC. These antibodies react with binding site determinants on anti-PC antibodies raised in all 15 inbred mouse strains tested regardless of histocompatibility or allotype, but fail to react with antibodies of other specificities or with anti-PC antibodies raised in other rodent species. These results correlate closely with other studies which show similar binding specificity of anti-PC antibodies raised in 17 different strains of mice. The site-associated idiotypic determinant(s) is clearly distinct from that detected by mouse anti-HOPC 8 antisera. This latter determinant(s) is present on anti-PC antibodies of only a few strains of mice and may not be in the binding site.

Clonal nature of the immune response to phosphorylcholine. I. Specificity, class, and idiotype of phosphorylcholine-binding receptors on lymphoid cells.

The relationship between receptor molecules on antigen-binding lymphocytes (ABC) and antibody produced by antibody-secreting cells was studied in inbred strains of mice using the immune response to phosphorylcholine (PC) as a model system. Splenic and lymph node lymphocytes of nonimmune mice possess rare lymphocytes which bind (125)I-labeled PC-bovine serum albumin. The frequency of PC-ABC increases after immunization and is paralleled by a rise in the frequency of PC-specific antibody-producing cells. Both of these responses are thymus independent. The receptors on these ABC display specificity for PC and are exclusively of the IgM class. In one of the strains, BALB/c, the receptors possess the same idiotype and fine degree of specificity for PC and two of its analogues, glycerophosphorylcholine and choline, that are characteristic of a PC-binding myeloma, HOPC 8. Furthermore, the idiotype and class of the receptor in these mice do not change during the course of the immune response. These data provide more direct evidence for the immunelogic relevance of receptor-bearing lymphocytes.

Clonal nature of the immune response. II. The effect of immunization on clonal commitment.

An inbred strain can produce several hundred different anti-group A carbohydrate (GAC) antibodies, as analyzed by isoelectric focusing. However, each individual mouse produces the bulk of its anti-GAC antibody as only one or two different spectrotypes, which appear to be randomly chosen. By using adoptive transfer techniques, we have observed that clonal commitment occurs very early in immunization, sometimes even before immunization, and thus does not result from competition among B cells for antigen. X

Clonal dominance. I. Restricted nature of the IgM antibody response to group A streptococcal carbohydrate in mice.

The IgM antibody response of mice to the streptococcal group A carbohydrate (GAC) was measured. With most strains tested, large amounts of IgM antibody were produced; in AKR mice, over 1% of the total nucleated spleen cells secreted IgM anti-GAC antibody after hyperimmunization. The relative avidity of the antibody was extimated by a modification of the Jerne plaque assay where spleen cells from individual mice were tested against erythrocytes with varying GAC epitope densitymthese studies showed that the earliest, as well as latest, IgM antibodies produced were highly restricted in avidity heterogeneity. No evidence of affinity maturation was seen upon hyperimmunization. These data favor the conclusion that the restricted IgG response seen in mice hyperimmunized to GAC is not the result of affinity driven competition for antigen among precursor cells.

Antigen mediation of a late-acting suppressor T-cell activity.

Carrier-specific suppressor T cells can suppress antibody secretion by high avidity IgG plaque-forming cells (PFC) within 90 min in vitro. This process can be blocked by the inclusion of soluble carrier in the cell mixture or by the exposure of target cells to anti-carrier antibodies or pronase. Moreover, suppression can be augmented by PFC exposure to the soluble hapten-carrier conjugate. Finally, carrier specificity may be overcome by preincubation of the target population with a hapten-heterologous carrier before addition of heterologous carrier ATC. Thus, it is likely that high avidity suppression depends upon immunogen bound to the surfaces of antibody-secreting cells which serves as a target for suppressor cells or molecules.

Receptors on immunocompetent cells. IV. Direct measurement of avidity of cell receptors and cooperative binding of multivalent ligands.

The interaction of antigen with specific, cell-associated receptors was measured in thermodynamic terms. The binding of (125)I-labeled 2,4-dinitrophenyl guinea pig albumin (DNP(16)GPA-(125)I) to lymphocytes from guinea pigs immunized to DNP(16)GPA is a temperature-dependent, reversible process. Measurement of association and dissociation rates of antigen-receptor complexes permits calculation of antigen-cell binding constants. These may also be calculated by equilibrium-binding techniques. Although differences in the constants calculated in these two ways exist, a clear increase in avidity of cell receptor for antigen occurs in the course of the immune response. This change in receptor avidity provides evidence that the time-dependent change in affinity of serum antibody (maturation) indeed has a cellular basis. The magnitude of the equilibrium constant is, in part, due to binding of more than one DNP group per molecule of antigen. Thus, multivalent ligands bind more effectively to cell receptors than univalent or paucivalent ligands when measured by the number of antigen molecules bound, the dissociation rate of antigen-receptor complexes, and in the relative capacity to inhibit a standard multivalent ligand (DNP(16)GPA-(125)I) from binding.

Receptors on immunocompetent cells. V. Cellular correlates of the "maturation" of the immune response.

During the course of the immune response to dinitrophenylated guinea pig albumin (DNP-GPA), a striking and parallel increase in avidity for epsilon-DNP-L-lysine occurs in the receptors on antigen-binding lymphocytes, antibody secreted by individual plaque-forming cells, and serum antibody molecules. A detailed analysis of the avidity distribution of antibody produced by plaque-forming cells indicates that this "immunologic maturation" is primarily due to a preservation of the high avidity subpopulation and a striking loss in the low avidity population rather than to sequential appearance of these cells. Moreover, the demonstration of the increased avidity of receptors of antigen-binding lymphocytes, which appear to be precursors of antibody-synthesizing cells, strongly suggests that the antigen-driven selectional process operates primarily on this cell type.

Receptors on immunocompetent cells. II. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding lymphocytes of normal guinea pigs.

Nonimmunized guinea pigs possess rare lymphocytes which bind sufficient 2,4-dinitrophenyl-guinea pig albumin-(125)I (DNP-GPA) to their surface to be detected by short-term radioautography. The cells occur in the lymph nodes, spleen, peripheral blood, and bone marrow with a frequency of approximately 40/100,000 lymphocytes, but are absent from the thymus. The receptors of these cells are largely specific for the haptenic group (epsilon-DNP-L-lysine) as shown by inhibition of DNP-GPA-(125)I binding with epsilon-DNP-L-lysine and with DNP bovine serum albumin (DNP-BSA). Furthermore, these cells specifically adsorb to agarose beads to which either DNP-GPA, DNP-BSA, or DNP-keyhole limpet hemocyanin (KLH) has been covalently linked. This hapten specific depletion of DNP-GPA-(125)I antigen-binding cells (ABC) correlates with a similar diminution in the capacity of adsorbed populations to transfer primary responsiveness to DNP-KLH to irradiated syngeneic recipients. Fluoresceinated anti-immunoglobulin binds to the surface of some guinea pig lymphocytes, and all DNP-GPA-(125)I ABC, as shown by a double-label technique. The great majority of DNP-GPA ABC and human gamma-globulin ABC possess surface Ig molecules of the gamma(2) heavy chain class. Preincubation of cell suspensions with anti-gamma(2) antibody markedly diminishes the number of DNP-GPA-(125)I ABC which are detected, strongly suggesting that the receptors of these cells are immunoglobulin molecules, most of which possess gamma(2) heavy chains. The specificity characteristics of DNP-GPA-(125)I ABC are strikingly different from those of cells mediating a cellular immune response to DNP-GPA, indicating major differences in the specificity and nature of the receptors of these cell types.

Polymorphism of Fc receptor on murine B cells is Igh-linked.

Analysis of mouse IgG binding to Fc receptors on mouse B cells indicates that the IgG1, IgG2a, and IgGb subclasses bind to the same receptor. No differences in affinity were detected among subclass or between mouse strains. This same receptor bound rat IgG with an affinity that differed between mouse strains. This polymorphism in affinity for rat IgG maps to chromosome 12 distal to the Igh locus.

Structural, functional, and idiotypic characteristics of a phosphorylcholine-binding IgA myeloma protein of C57BL/ka allotype.

An IgA phosphorylcholine (PC)-binding myeloma protein with IgCH allotypic determinants different from those of BALB/c mice is characterized. The myeloma, CBPC 2, was induced in the CB-20 strain of mice which is congenic to BALB/c but differs from it by carrying the A15 allotypic determinant of C57BL/ka mice. Sequence analysis of the CBPC 2 light chain through the first hypervariable region, as well as isoelectric point analysis, show that this chain is indistinguishable from that of T15, a PC-binding myeloma protein of BALB/c origin. The heavy chains of CBPC 2 and T15 differ by only two amino acids (positions 14 and 16) through the first hypervariable region. As measured by inhibition of precipitation, both CBPC 2 and T15 have the same specificity for PC, glycerophosphorylcholine, acetylcholine, and choline. In addition, CBPC 2 possesses the binding site-associated idiotypic determinant which is present on T15. However, like normal or induced C57BL/6 anti-PC antibody, it does not possess the nonbinding site idiotypic determinant.

Carrier function in anti-hapten antibody responses. IV. Experimental conditions for the induction of hapten-specific tolerance or for the stimulation of anti-hapten anamnestic responses by "nonimmunogenic" hapten-polypeptide conjugates.

Administration of nonimmunogenic 2,4-dinitrophenyl (DNP) conjugates of copolymers of D or L-glutamic acid and lysine (GL) induces hapten-specific tolerance in nonimmune and DNP-ovalbumin-primed strain 13 guinea pigs. This tolerant state is evidenced by depressed anti-DNP antibody synthesis in response to challenge with DNP-ovalbumin and by a diminished frequency of DNP-specific antigen-binding cells and of anti-DNP antibody-secreting cells. Such a nonimmunogenic compound (DNP-D-GL) will nevertheless elicit a DNP-specific anamnestic antibody response when administered at an appropriate time to DNP-ovalbumin-primed guinea pigs undergoing a graft-versus-host reaction. These experiments are discussed in terms of a two-cell theory of stimulation of antibody responses.

Subclass restriction of murine antibodies. II. The IgG plaque-forming cell response to thymus-independent type 1 and type 2 antigens in normal mice and mice expressing an X-linked immunodeficiency.

Antigens have been classified previously into three categories, thymus-dependent (TD), thymus-independent type (TI) 1, and TI-2, based upon thymic dependence and ability to stimulate an immunodeficient strain of mouse, CBA/N. Here we demonstrate that the different antigen classes elicit IgG antibodies of different subclasses. TD antigens stimulate predominantly IgG1 antibodies, with smaller amounts of IgG2 and IgG3 being expressed. TI-1 antigens stimulate almost no IgG1 antibodies and equal amounts of IgG2 and IgG3. TI-2 antigens elicit predominantly IgG3 antibodies. Mice expressing the CBA/N phenotype are known to be nonresponsive to TI-2 antigens. This was confirmed in this study. In addition, we demonstrate that the IgG3 component of the response to TI-1 antigens is virtually absent in mice expressing the CBA/N phenotype, which supports our previous finding that the CBA/N defect may be restricted to a B-lymphocyte subpopulation containing most of the precursors of IgG3-secreting cells.

Structural correlates of cross-reactive and individual idiotypic determinants on murine antibodies to alpha-(1 leads to 3) dextran.

For the first time V-region amino acid sequence differences have been correlated with the expression of cross-reactive and individual idiotypes through an analysis of 12 dextran-binding proteins. This correlation has been possible because of the apparent sequence identity of the corresponding lambda chains. Expression of a cross-reactive idiotype was localized to two residues and/or a carbohydrate in the second hypervariable region of the heavy chain. Two individual idiotypes correlate with the two amino acids within the third hypervariable region that comprises the D segment of the dextran-binding proteins. These results demonstrate that idiotype reagents can recognize two amino acid differences within V and D segments of classical variable regions. In anti-dextran antibodies, cross-reactive idiotypes involve V-region determinants, whereas individual idiotype determinants correlate with D-segment variation.

Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.

Hapten-specific tolerance. Preferential depression of the high affinity antibody response.

The induction of tolerance in guinea pigs with a 2,4-dinitrophenyl (DNP) derivative of a copolymer of copolymer of D-glutamic acid and D-lysine (D-GL) leads to a preferential depression of the capacity to produce high affinity anti-DNP antibody in response to immunization with DNP-guinea pig albumin. Thus, immunization 2 wk after tolerance induction with 3 mg of DNP-D-GL results in an immune response in which individual plaque-forming cells (PFC) secreting high affinity anti-DNP antibody are absent and in which the affinity of circulating anti-DNP antibody is reduced. A similar, but less marked, suppression is seen when 0.3 mg of DNP-D-GL is used for tolerance induction. If immunization is delayed until 2 months after tolerance induction, then suppression is restricted to the highest avidity PFC group. Our data is consistent with a state of tolerance in the pool of precursors of anti-DNP antibody-secreting cells induced as a result of their interaction with DNP-D-GL in the absence of specific "helper" cells, which appear to be lacking for DNP-D-GL. In such a situation, the affinity of receptors on precursor cells for tolerogen and the concentration of tolerogen appear to be crucial determinants of whether an individual cell will become tolerant.

Deletion of b5 immunoglobulin-bearing lymphocytes in allotype-suppressed rabbits.

The proportion of peripheral blood lymphocytes with cell surface b5 immunoglobulin (Ig) was compared with serum b5 Ig levels in allotype-suppressed and recovering b5 homozygous rabbits in order to establish the cellular defect in suppression and to determine the relation between lymphocyte membrane Ig and secreted Ig of the same allotype. In rabbits fully suppressed for b5, the absence of circulating b5 Ig is reflected in a complete deletion of b5-bearing lymphocytes. During spontaneous escape from suppression, the appearance of b5-bearing lymphocytes precedes the appearance of detectable serum b5. During recovery from suppression b5-bearing lymphocytes recover rapidly toward normal levels, but circulating b5 levels remain chronically and disproportionately depressed.

Receptors on immunocompetent cells. 3. Specificity and nature of receptors on dinitrophenylated guinea pig albumin- 125 I-binding cells of immunized guinea pigs.

Guinea pigs immunized with 2,4-dinitrophenyl-guinea pig albumin (DNP-GPA) possess lymphocytes which specifically bind sufficient DNP-GPA-(125)I to their surface to be detected by radioautography. These lymphocytes are present in the draining lymph nodes in a frequency of approximately 50/1000 lymphocytes in animals immunized 2-4 wk earlier with DNP-GPA in complete Freund's adjuvant. Nonimmunized animals have approximately 0.4 DNP-GPA antigen-binding cells (ABC) per 1000 lymphocytes. An increase in the frequency of DNP-GPA ABC in peripheral blood is detectable by 5 days after immunization, which is before the time that serum anti-DNP antibody is measurable. The receptors of these ABC are hapten specific in that free epsilon-DNP-L-lysine, at low concentration, inhibits the binding of DNP-GPA-(125)I; DNP bovine serum alumbin (DNP-BSA) is equivalent to DNP-GPA in the inhibition of binding of DNP-GPA-(125)I to ABC; and both DNP-GPA agarose beads and DNP-BSA agarose beads specifically adsorb DNP-GPA-(125)I ABC. Anti-immunoglobulin antisera, particularly anti-gamma(2) sera, inhibit the binding of DNP-GPA-(125)I to these cells implying that the receptors are immunoglobulin, primarily of the gamma(2) heavy chain class. DNP-GPA-(125)I ABC appear to represent precursors of antibody-secreting cells and have specificity characteristics which are very different from cells, of similarly immunized guinea pigs, which mediate a cellular immune response to DNP-GPA.