Paul D. Gottlieb

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10−6 or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1∶2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes.In addition, three new strains of mice differing from existing strains in the region of theLyt-2 and4Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearingLyt-2a andLyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b, congenic with AKR/J and bearing theLyt-2b andLyt-3b alleles of Balb/cJ.

Immunoglobulin classes and subclasses in alloantisera to mouse thymocyte surface antigens

A variety of cytotoxic alloantisera directed against murine cell surface components were analyzed for their content of specific IgG1, IgG2a, and IgG2b antibodies by radioimmunoassay, and specific IgM antibodies by 2-mercaptoethanol inhibition of cytotoxicity. Results indicate: (1) that the IgM and IgG subclass content of specific antibodies in anti-Lyt antisera produced at M.I.T. and Sloan-Kettering using the same donor and recipient strains are similar; (2) that specific antibodies produced concurrently in the same mice against at least two different alloantigens (Lyt-3.1 and H-2k) can differ in IgG subclass content; and (3) that preparation of alloantisera against a given antigen (Lyt-3.1) using different combinations of donor and recipient strains can yield specific antibodies which differ in IgG subclass and cytotoxic IgM content. Thus, it appears that humoral immune responses to cell surface alloantigens are not random, but reflect both the antigens being recognized and the strains employed for the immunization.

Genetic and Structural Studies of a V-Region Marker in Mouse Immunoglobulin Light Chains

This chapter describes the characteristics and strain distribution of a genetic marker discovered by Edelman and Gottlieb (1970) in the V regions of serum Ig L chains of unimmunized mice. Gottlieb (1974) demonstrated that expression of this V-region marker (called the IB-peptide marker) is closely linked to the Ly-3 genetic locus governing expression of a thymocyte surface antigen (Boyse et al., 1971). The Ly-3 locus resides in linkage group XI on chromosome 6 of the mouse (Itakura et al., 1972). If the locus governing expression of the IB-peptide marker is a structural locus for L-chain V regions, then it too resides in linkage group XI, and this would be the first instance in any species in which structural genes for an Ig chain have been placed in a particular linkage group. The possibility exists, however, that the locus which maps in linkage group XI does not contain structural genes for light-chain V regions, but rather is a regulatory locus which governs the expression of structural genes encoded in this linkage group or elsewhere in the genome.

ROLE OF THE LIGHT CHAIN IN STUDIES OF LINKAGE OF GENES CONTROLLING IDIOTYPE AND HEAVY CHAIN ALLOTYPE

Publisher Summary This chapter presents a study whose purpose was to determine whether the linkage between idiotype and allotype would still obtain when the second strain used in breeding studies was one of the four strains possessing the unique VK marker. Two such strains have now been investigated: PL/J and C58/J. The data obtained with strain PL mice are reviewed in the chapter together with data in which strain C58/J was used as the second strain in breeding studies with A/J mice. The chapter presents some results obtained with offsprings of F1 mice backcrossed to the corresponding non-A/J strains. In all cases, offsprings lacking the A/J allotype failed to produce significant amounts of the cross-reactive idiotype, and l000 ng gave less than 20% inhibition in the radioimmunoassay in each case. By contrast, all mice that were A/J aliotype–positive produced a significant amount of the cross-reactive idiotype, although in two mice, the values were relatively low.

The ϕ6 Cystovirus Protein P7 Becomes Accessible to Antibodies in the Transcribing Nucleocapsid: A Probe for Viral Structural Elements

Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the five-fold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe- reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of Vκ and VH genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein’s antigenic sites.

Alterations in expression of murine thymocyte proteins and glycoproteins during fetal thymus development.

To describe the biochemical alterations that accompany T cell differentiation and proliferation in the mouse thymus, proteins and Con A-binding glycoproteins of adult thymocytes and of fetal thymocytes on days 14–19 of gestation were compared. Cells were labelled with 125I and lactoperoxidase, NP-40 extracts were prepared, and both whole extracts and immunoprecipitates obtained with rabbit anti-mouse thymocyte serum and Staphylococcus aureus, Cowan I strain, were subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) under reducing conditions. In addition, NP-40 extracts of unlabelled fetal and adult thymocytes were subjected to SDS-PAGE, and the gels reacted with 125I-labelled Con A before drying and autoradiography. Results obtained suggest that: (1) T200-like components of early fetal thymocytes have a higher apparent mol. wt(200+ K) than those of late fetal and adult cells; (2) the quantity of the T25 component which bears the Thy-1 alloantigen increases from very low early in gestation to adult levels by day 17–19; (3) adult thymocytes express a Con A-unreactive surface component of apparent mol. wt 12,000 which is present in much lower amounts on fetal thymocytes; (4) fetal thymocytes on all days of gestation express a Con A-unreactive surface component of apparent mol. wt 10,000 which is barely (if at all) detectable on adult thymocytes.

Control of the antibody response of C57BL/6 Lyt-congenic strains to the Lyt-3.1 alloantigen by a gene linked to theLyt-2 locus

Congenic anti-Lyt-3.1 sera have recently been produced by immunizing B6-Lyt-2a mice with thymocytes from either B6-Lyt-2a, Lyt-3a or B6-Lyt-2a, Lyt-3a, H-2k mice (Boos et al. 1978). Surprisingly, mice of the congenic strain B6 failed to produce either anti-Lyt-2.1 or anti-Lyt-3.1 cytotoxic antibodies after identical immunizations. To determine the genetic basis for the difference in response to Lyt-3.1, (B6 × B6-Lyt-2a)Fa mice and progeny of the backcross, (B6 × B6-Lyt-2a)F1 × B6-Lyt-2a, were immunized with B6-Lyt-2a, Lyt-3a, H-2k thymocytes. In addition, thymic biopsies of backcross progeny were performed and thymocytes tested for the Lyt-2.2 antigenic specificity. Results indicate that gene(s) governing the immune response to Lyt-3.1 is (are) linked to theLyt-2 locus, and that the responder allele (linked toLyt-2a) shows very poor penetrance in Lyt-2a/Lyt-2b mice.

[59] Lyt-1, Lyt-2, and Lyt-3 antigens

Publisher Summary This chapter explores that the Lyt-1, Lyt-2, and Lyt-3 alloantigens resides on the surface of mouse thymocytes and on subpopulations of peripheral lymphocytes. It discusses that the Lyt-2 and Lyt-3 cell surface molecules has been kindled by the finding that they are T cell specific, and they mark certain functional subpopulations of T cells. The Lyt-2 and Lyt-3 alloantigens are present on cytotoxic killer and suppressor T cells but not on T cells which mediate helper function or delayed type hypersensitivity (DTH). The chapter also reviews that the presence of Lyt-2 and Lyt-3 on only certain functional subsets of T cells has raised the possibility that they may participate in the specialized function of the cells that bear them. Close linkage of Lyt-2 and Lyt-3 to the immunoglobulin light chain locus raised the possibility that these molecules might be structurally related to immunoglobulins and might contribute in some way to the specific antigen receptor on these T cells.

Digestion of a mouse λ1 chain with staphylococcal protease yields a large V-region fragment accessible to direct amino acid sequence analysis

Digestion of the MOPC 104E mouse λ1 chain with staphylococcal protease under conditions reported to restrict proteolytic cleavage to glutamic acid residues has generated a large fragment of the V-region which is accessible to automated amino-acid sequence analysis. The forty residues of amino-acid sequence obtained were consistent with the published sequence (Kabat et al., 1976) with the exception that cleavage carboxyterminal to position 7 suggests that this residue is glutamic acid, rather than glutamtne as published. Since the positions of glutamic acid in murine λ1 and λ2 chains are highly conserved, it is likely that the method described will be applicable to most, if not all, λ1 chains and possibly λ2 chains as well.