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

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

Antibodies to idiotypes of isologous immunoglobulins.

To determine if the immunoglobulins (Igs) capable of eliciting the formation of isologous anti-idiotypic antibodies are rare exceptions, BABL/c mice were immunized with five myeloma proteins of BALB/c origin. Anti-idiotypes were produced against all but one. The idiotype of the exception (T15) is remarkably abundant in BALB/c mice, whose unresponsiveness is probably due to tolerance. Nevertheless, prolonged immunization with T15 finally induced the formation of isologous antibodies that seemed largely to be specific for IgA proteins, especially those with k-light-chains; the reactions of a few of these isologous antisera with T15 were slightly inhibited by phosphorylcholine, suggesting that some anti-idiotypes were probably formed even to this unusually prevalent idiotype. It is likelythat under appropriate conditions almost any myeloma protein can elicit isologous anti-idiotypes.

The strange cross-reaction of menadione (vitamin K3) and 2,4-dinitrophenyl ligands with a myeloma protein and some conventional antibodies.

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of [(3)H]-epsilon-DNP-L-lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K(3)). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 10(5) L/M (at 4 degrees C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal).

Sequential changes in the relative affinity of antibodies synthesized during the immune response.

The anti-2,4-dinitrophenyl (DNP) antibodies synthesized by suspensions of lymph node cells obtained at various intervals from rabbits that had been immunized with DNP-bovine gamma-globulin increased progressively in their affinity for the dinitrophenyl determinant. This change accompanied and was apparently responsible for a similar change in the binding properties of anti-DNP antibodies isolated from the serum. The rate of change in affinity was related to the dose of immunogen: increasing the dose delayed the change. The antibodies formed during a brief (5 hr) incubation in vitro were heterogeneous in their binding properties. Therefore, the mixing in the circulation of molecules synthesized at different times may contribute to, but is not alone responsible for, the heterogeneity in the serum antibodies. Variability in binding did not appear to be related to heterogeneity in immunoglobulin class. Indeed, the variations in relative affinity occurred entirely within the gammaG-immunoglobulins.

The relative affinity of antibodies synthesized in the secondary response.

In response to a second injection of rabbits with a dinitrophenylated antigen, given 2 months to 2 yr after the first injection, there was rapid synthesis of large amounts of antibody high in relative affinity for the dinitrophenyl (DNP) determinant. The antibodies formed 3 days after restimulation were already high in affinity. Amounts of antigen too small to elicit detectable antibody production may prime the animal for a partial secondary response characterized by the formation of antibody of intermediate affinity after a second antigenic stimulus. Investigations into the specificity requirements for the secondary response indicated that variation in the carrier protein and in the haptenic determinant could be tolerated. Thus, after immunization with DNP-bovine gamma-globulin, DNP-hemocyanin elicited the vigorous production of high affinity anti-DNP antibodies. However, DNP serum albumin was much less effective: it elicited a secondary response in some animals primed with DNP-bovine gamma-globulin only when the interval between injections was increased from 10 to 28 wk. A secondary response was also evoked when the haptenic determinent of the second immunogen differed slightly from that of the one injected initially (i.e., 2,4,6-trinitrophenyl versus 2,4-dinitrophenyl).

Binding of monomeric immunoglobulins to Fc receptors of mouse macrophages.

The binding properties of surface receptors of immunoglobulins on mouse macrophages were studied with mouse myeloma proteins and normal peritoneal macrophages, thioglycollate-stimulated macrophages, and a macrophage cell line, P388D1. Primary cultures of mouse embryo fibroblasts served as controls. IgG2a proteins were bound strongly;IgG2b was bound weakly (one-twentieth as well as IgG2a);IgM, IgA, and IgG1 were not bound significantly. The number of binding sites per cell for IgG2a was 4 X 10(5) for thioglycollate-stimulated cells and 1 X 10(5) for normal and P388D1 cells. Binding was exothermal: with decreasing temperature the equilibrium (association) constants increased and dissociation rate constants decreased (at 37degreesC the respective values were 2 X 10(7) M-1 and 0.26 min-1, the latter value corresponds to a half time for dissociation of 2.6 min). From the rapidity of association and dissociation, it appears that the surface of the macrophage is in a dynamic equilibrium with IgG2a molecules in the cell's immediate microenvironment. The receptors for IgG2a are clearly specific for determinants in the immunoglobulin constant domain: two IgG2a proteins with greatly different isoelectric points (determined by isoelectric focusing) were bound with the same affinity to the same receptors; moreover, the Fc fragment was bound and Fab fragments were not. The Fc receptors for IgG2a proteins were readily eliminated by exposing macrophages briefly to trypsin. The receptors were regenerated during subsequent cultivation in serum-free medium; regeneration was inhibited totally by cycloheximide and partially by actinomycin D.

Specificity of the immune response to the 2,4-dinitrophenyl and 2,4,6-trinitrophenyl groups. Ligand binding and fluorescence properties of cross-reacting antibodies.

THE PRINCIPAL RESULTS OF THE PRESENT STUDY ARE: (a) the failure to find an antibody subset that binds a cross-reacting ligand better than the comparable homologue in spite of the use of isolation methods that select for such antibody molecules; (b) the isolation of antibody subsets with virtually indistinguishable average intrinsic association constants for homologous and cross-reacting ligands, but which nevertheless have physical properties (Qmax and relative fluorescence coefficient) that readily distinguish these subsets according to their origin in response to antigenic stimulation with DNP- or with TNP-protein; (c) the demonstration, by precipitin reaction and measurement of association constants for homologous and cross-reacting haptens, of generally greater cross-reactivity among high affinity anti-DNP and anti-TNP antibodies, i.e. low affinity antibodies are generally more discriminating; (d) the selection of anti-DNP and anti-TNP antibody subsets that are distinctive in their ability to show spur formation in gel diffusion reactions with homologous and cross-reacting antigens. These results suggest that in the initial cellular response to antigenic stimulation, DNP-BgammaG and TNP-BgammaG stimulate virtually nonoverlapping sets of antigen-sensitive cells, despite the great similarity of these two immunogens. With prolonged stimulation this specificity wanes, giving rise to a more degenerate response evident in the greater cross-reactivity of the antibodies produced later in immunization.

An assay for determining the relative affinity of trace amounts of labeled antibodies.

All assay for the binding efficiency or "relative affinity" of trace amounts of radioactively labeled anti-2,4-dinitrophenyl (DNP) antibodies has been developed. The assay measures the relative ability of the labeled antibodies to combine and precipitate with antigen in the presence of a large amount of unlabeled reference antibody of the same specificity. It has been possible to correlate the relative affinity of the labeled antibodies for dinitrophenylated antigens with the association constants of the reference antibodies for simple univalent DNP ligands.

Expression of structurally diverse Qa-2-encoded molecules on the surface of cloned cytotoxic T lymphocytes.

Extracts of 125I-labeled cloned murine cytotoxic T lymphocytes (CTL) were immunoprecipitated with alloantisera to the cloned CTL and rabbit antisera to beta-2 microglobulin. Polyacrylamide gel electrophoresis (PAGE) of the specific precipitates revealed, as expected, 125I-labeled components that corresponded to products of class I genes of the major histocompatibility complex (MHC). However, additional class I gene products of relatively low apparent molecular weight (Mr) were also observed. Similar analyses of spleen cells from a variety of MHC-congenic mouse strains suggested that the class I molecules of relatively low Mr are encoded in the Qa-2 region of the MHC, and this was confirmed by immunoprecipitation with a monoclonal antibody to Qa-2. Surprisingly, however, the cell surface Qa-2 molecules of different CTL clones differed in Mr, in isoelectric focusing (IEF) pattern, and in the number of distinguishable molecules expressed per clone: some clones seemed to express only a single Qa-2-encoded molecule while others expressed two distinct ones. Treatment of the immunoprecipitated Qa-2 with endoglycosidase F (Endo F) resulted in a decrease in Mr of approximately 5,000-6,000, corresponding to the expected loss of N-linked oligosaccharides, but the decrease did not eliminate structural variability among the clones. Structural diversity of the Qa-2-encoded molecules expressed on CTL could arise because CTL clones differ (a) in the particular Qa-2 genes they express, (b) in the way they splice Qa-2 gene transcripts or, perhaps, (c) in Endo F-resistant oligosaccharides on their Qa-2 molecules.

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes.

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular mass; dot blot hybridization with synthetic oligonucleotides corresponding to the active sites of two known murine CTL esterases suggests homology to the murine enzyme HF. However, serine esterase was present at only approximately 10% of the level found in murine CTLs, and was not secreted during CTL-target cell interaction; moreover, hemolytic activity could not be detected in any of the seven cell lines tested. The results suggest that the human CTLs examined here kill their target cells by a mechanism different from that used by most cloned murine CTLs.

Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes.

To investigate the destruction of target cells by murine CTLs, we examined intracellular Ca2+ concentrations ([Ca2+]i) and DNA fragmentation in target cells. Changes in [Ca2+]i were followed by flow cytometry by loading the cells with indo-1, a Ca2+-binding fluorescent dye, and determining the ration of fluorescence intensities at 405 nm (emission maximum for Ca2+-bound dye) over 480 nm (emission maximum for the free dye). Within minutes after interacting with the cytolytic granule fraction that had been isolated from CTLs, [Ca2+]i in target cells was strikingly increased. A pronounced increase in [Ca2+]i was also observed in target cells when they were specifically recognized by intact CTLs. Since ionomycin, a Ca2+ ionophore, caused a similar increase in [Ca2+]i and lysed cells (provided that extracellular Ca2+ was present), it appears that a sustained high level of [Ca2+]i is cytolytic. In contrast with other cells, CTLs, which have been shown to be refractory to granule-mediated lysis and to be poor targets for other CTLs, did not manifest an elevation in [Ca2+]i when they were similarly loaded with indo-1 and treated with isolated granules. The characteristic cleavage of target cell DNA into nucleosome-sized fragments was also induced by isolated granules as well as by valinomycin, a K+ ionophore, but not by ionomycin. The results support the view that lysis of most target cells by cloned CTLs is due primarily to target cell membrane changes that are fundamentally equivalent to the formation of nonspecific ion channels. The resulting large increase in [Ca2+]i is probably responsible for target cell lysis; and changes in intracellular ion concentrations also appear to be responsible for DNA fragmentation, probably by activating endogenous target cell endonucleases.

Resistance of cytotoxic T lymphocytes to the lytic effects of their toxic granules.

A cytotoxic T lymphocyte (CTL) characteristically kills target cells one after the other by releasing toxic granules that contain one or more cytolytic components. To determine how CTLs avoid destroying themselves when they release granules and lyse target cells, 7 murine CD8+ CTL cell lines were compared with 19 other cell lines for susceptibility to lysis by the isolated toxic granules. Murine CD8+ CTLs were clearly the most resistant cells: granules did not lyse them even after they were exposed to azide, cyanide, and 2-deoxyglucose, conditions that were found to enhance the susceptibility of all the other cells tested, including other T cells. Thus, resistance of CD8+ CTLs to cytotoxic granules appears to be independent of cellular ATP. To reconcile these findings with other observations that, under some circumstances, CTLs can be lysed by other CTLs, we suggest a model in which a CTL releases only a limited proportion of its toxic granules at each antigen-specific encounter with a target cell; the amount released is sufficient to kill most target cells but to leave the CTL undamaged and with enough granules to attack other target cells.

Homeostasis-stimulated proliferation drives naive T cells to differentiate directly into memory T cells.

The developmental requirements for immunological memory, a central feature of adaptive immune responses, is largely obscure. We show that as naive CD8 T cells undergo homeostasis-driven proliferation in lymphopenic mice in the absence of overt antigenic stimulation, they progressively acquire phenotypic and functional characteristics of antigen-induced memory CD8 T cells. Thus, the homeostasis-induced memory CD8 T cells express typical memory cell markers, lyse target cells directly in vitro and in vivo, respond to lower doses of antigen than naive cells, and secrete interferon gamma faster upon restimulation. Like antigen-induced memory T cell differentiation, the homeostasis-driven process requires T cell proliferation and, initially, the presence of appropriate restricting major histocompatibility complexes, but it differs by occurring without effector cell formation and without requiring interleukin 2 or costimulation via CD28. These findings define repetitive cell division plus T cell receptor ligation as the basic requirements for naive to memory T cell differentiation.

In vivo cytotoxic T lymphocyte elicitation by mycobacterial heat shock protein 70 fusion proteins maps to a discrete domain and is CD4(+) T cell independent.

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8(+) cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4(+) T lymphocytes. We found that the ability to elicit CD8(+) CTLs depends on a discrete 200-amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8(+) T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8(+) CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4(+) T cell-deficient individuals.

Synthetic phospholipid vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxic T lymphocytes.

Synthetic phospholipid vesicles (liposomes) containing the purified glycoprotein (G) of vesicular stomatitis virus (VSV) and solubilized membrane proteins from cells of the appropriate H-2 haplotype elicited H-2-restricted cytotoxic T lymphocytes (CTL) that lysed VSV-infected target cells. The CTL were elicited by intact liposomes, not by released components. Thus, when spleen cells from VSV-primed H-2d X H-2b hybrid mice were stimulated with liposomes having G protein + membrane proteins from cells with one of the parental H-2 haplotypes, the resulting CTL lysed only VSV-infected target cells with that parent's H-2 type. This result argues against the view that T cells in general recognize only processed antigenic fragments on macrophages. Moreover, liposomes were only effective when G protein and cell membrane proteins were included in the same vesicles. This result suggests that for effective interaction with CTL precursors the antigen (G protein) and products of the H-2 complex must be closer to each other than 600--1,000 angstrom, the diameter of the lipid vesicles used in this study.

Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1.

We have established long-term cultures of several cell lines stably and uniformly expressing human immunodeficiency virus type 1 (HIV-1) in order to (a) identify naturally processed HIV-1 peptides recognized by cytotoxic T lymphocytes (CTL) from HIV-1-seropositive individuals and (b) consider the hypothesis that naturally occurring epitope densities on HIV-infected cells may limit their lysis by CTL. Each of two A2-restricted CD8+ CTL specific for HIV-1 gag or reverse transcriptase (RT) recognized a single naturally processed HIV-1 peptide in trifluoroacetic acid (TFA) extracts of infected cells: gag 77-85 (SLYNTVATL) or RT 476-484 (ILKEPVHGV). Both processed peptides match the synthetic peptides that are optimally active in cytotoxicity assays and have the consensus motif described for A2-associated peptides. Their abundances were approximately 400 and approximately 12 molecules per infected Jurkat-A2 cell, respectively. Other synthetic HIV-1 peptides active at subnanomolar concentrations were not present in infected cells. Except for the antigen processing mutant line T2, HIV-infected HLA-A2+ cell lines were specifically lysed by both A2-restricted CTL, although infected Jurkat-A2 cells were lysed more poorly by RT-specific CTL than by gag-specific CTL, suggesting that low cell surface density of a natural peptide may limit the effectiveness of some HIV-specific CTL despite their vigorous activity against synthetic peptide-treated target cells.

Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells.

Cytotoxic T lymphocytes can be activated by antibodies to their antigen-specific receptor complex (TCR-CD3) to destroy target cells, regardless of the specificity of the cytotoxic T cells. A novel hormone-antibody conjugate, consisting of an analog of melanocyte-stimulating hormone chemically coupled to a monoclonal antibody to CD3, the invariant component of the T cell receptor complex, was used to target human melanoma cells for destruction by human cytotoxic T lymphocytes that bear no specificity for the tumor cells. As targeting components of such anti-CD3 conjugates, hormones or growth factors are expected to prove more effective than antibodies to tumor-associated antigens in focusing the destructive activity of cytotoxic T cells on tumor target cells.

Tissue-specific expression of functionally rearranged lambda 1 Ig gene through a retrovirus vector.

To explore the potential use of retrovirus vectors for the transfer of genomic DNA sequences into mammalian cells, recombinant retroviral genomes were constructed that encode a functionally rearranged murine lambda 1 immunoglobulin gene. Several of these genomes could be transmitted intact to recipient cells by viral infection, although successful transmission depended both on the orientation of the lambda 1 sequences and on their specific placement within vector sequences. The lambda 1 gene transduced by viral infection was expressed in a cell lineage-specific manner, albeit at lower levels than endogenous lambda 1 gene expression in cells from the B-lymphocyte lineage. Vectors yielding integrated proviruses that lacked viral transcriptional enhancer sequences were used to show that neither viral transcription nor the viral transcriptional sequences themselves had any effect on the tissue specificity of lambda 1 gene expression or the absolute amount of lambda 1 transcription. Vector transcription did, however, dramatically decrease the amount of lambda 1 protein that could be detected in tranduced cells. These results suggest that retrovirus vectors may be useful reagents not only for the expression of complementary DNA sequences but also for studies of tissue-specific transcription in mammalian cells.

Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene.

Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.