The binding of antibody to the CD4 molecule inhibits mobilization of cytoplasmic free calcium ([Ca2+]i) in response to CD3 cross-linking on resting T cells. Similarly, when CD3 and CD4 are independently and simultaneously cross-linked, calcium mobilization is inhibited when compared to that induced by cross-linking CD3 alone. In contrast, when anti-CD4 and anti-CD3 are cross-linked together, calcium mobilization is substantially higher than from CD3 cross-linking alone. A heteroconjugate consisting of covalently bound CD3 and CD4 monoclonal antibodies (mAb) retains the ability to mobilize [Ca2+]i in CD4 cells at protein concentrations approximately two orders of magnitude lower than the free CD3 mAb, and the activity of the heteroconjugate is inhibitable by free CD4 mAb. The CD3/CD4 heteroconjugate also shows significantly greater activity in stimulation of inositol phosphate IP1, IP2 and IP3 synthesis in T cells than the CD3 mAb alone, and again the activity is inhibited by free CD4 mAb. The activity of the CD3/CD4 heteroconjugate is not simply due to oligomerization, since CD3/CD3 or CD4/CD4 homoconjugates or homoconjugate mixtures did not show increased activity. Other heteroconjugates (CD3/CD5 and CD3/CD28) were not different than the CD3/CD3 homoconjugate in their ability to increase [Ca2+]i. Purified CD4 T cells that do not respond to CD3 mAb in solution do respond to the CD3/CD4 heteroconjugate in solution by proliferating in the presence of a CD28 mAb, with a significant fraction of CD4 cells entering the second cycle within the first three days of stimulation. The CD3/CD4 heteroconjugate co-modulates the CD3 and CD4 receptors, indicating that the heteroconjugate is not simply anchoring the T cell receptor to the T cell surface like anti-CD3 on a solid surface. These results suggest that CD4 plays an active role in signal transduction when brought into close physical proximity to the CD3/T cell receptor complex during major histocompatibility complex class II-restricted antigen presentation.

Lyt-2 and Lyt-3 antigens are carried on separate disulfide-bonded subunits of the same cell surface macromolecules. These are present on thymocytes in a variety of multimeric forms consisting of disulfide-bonded dimers, tetramers, and hexamers of pairwise combinations of three subunits (30,000, 34,000, and 38,000 Mr). From reduced and alkylated Nonidet-P40 thymus extracts, a monoclonal anti-Lyt-3 precipitates only the 30,000 Mr subunit, but not the 30,000 Mr subunit. Almost all of the Lyt-2 and Lyt-3 subunits on the cell are covalently linked by disulfide bonds. However, small amounts of free Lyt-3 subunit was seen in some experiments. Similarly, small amounts of Lyt-2-3- material, consisting of dimers of the 38,000 and 34,000 Mr subunits were identified. Each of the three subunits migrated with a basic charge (pI greater than 8) on two-dimensional gels. Cytotoxic effector cells that are blocked by anti-Lyt-2 and anti-3 can be treated with trypsin and other arginine-specific proteases to remove these antigens. At low concentrations of these proteases, Lyt-3 antigens are selectively removed. After selective removal of Lyt-3 antigens, cytotoxic effector cells are still active and blocking of activity by anti-Lyt-3 is significantly reduced, whereas blocking of activity by anti-Lyt-2 is significantly increased. Neither Lyt-2 nor Lyt-3 is allelically excluded on thymocytes or T cells. These results suggested that the Lyt-2, Lyt-3 macromolecules are associated with but do not serve as the T cell antigen receptor.

Memory B cells which give rise to IgG antibody-producing cells were generally assumed to be IgG-bearing cells. However, recent studies indicating that very few IgG-bearing cells exist in lymphoid tissue brought this assumption into question. In this study, we examined directly the question whether IgG-bearing cells contain functional precursors of IgG antibody-producing cells. Using the adoptive secondary immune response, we demonstrated that Ig-1 b-bearing cells, isolated with the fluorescence-activated cell sorter (FACS), are the functional precursors of Ig- 1 b-producing cells. Further, we have enriched IgG2 and IgG1 memory B cells using the FACS. The results show that IgG2-bearing cells are the functional precursors of the IgG2 antibody-producing cells. Likewise, the IgG1-bearing cells are the functional precursors of IgG1 antibody-producing cells. Thus, IgG memory cells have surface IgG which indicates the class and allotype commitment of the memory cell and its progeny antibody-forming cells.

No abstract

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.