IgD receptor-mediated signal transduction in T cells.

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.

Human T cell IgD receptors react with O-glycans on both human IgD and IgA1.

Previous studies on murine T cell IgD-R have shown that these receptors recognize N-glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD-R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD-coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, -2, -3, -4, IgA2, IgE and IgM fail to inhibit significantly at 20 microg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 microg and 10 microg/assay. Human IgA1 and IgD both contain Gal-1 --> 3-GalNac-rich O-linked glycans, and on this basis are both bound to ricin and jacalin. The O-linked glycans may therefore also represent the common moiety binding to IgD-R. Disaccharides Gal-1 --> 3-GalNac, and Gal-1 --> 4-Glc at 10 microg/assay blocked IgD rosetting while Gal-1 --> 6-Glc did not. We conclude that the human IgD-R is a lectin, differing from the murine IgD-R in that it has both IgA1 and IgD as ligands.

Inhibition of sphingolipid synthesis down-modulates CD4 expression by peripheral blood T lymphocytes and T lymphoma cells.

Recent studies have demonstrated that L-cycloserine, an inhibitor of sphingolipid biosynthesis, interferes with the life cycle of HIV. Experiments with human T cells and CD4+ T lymphoma cells were performed to examine possible mechanisms. L-CS selectively down-modulated CD4 expression without affecting the expression of CD3 and CD8. L-cycloserine also inhibited T cell mitogen responses without affecting IL-2 production. Membranes prepared from L-CS-treated T lymphoma cells showed changes in lipid composition that correlated with changes in membrane microviscosity. These results suggest that normal expression of CD4 may depend upon sphingolipid biosynthesis in contrast with the other CD determinants measured. Selective inhibition of CD4 by L-cycloserine together with its antiviral effects may offer a novel approach for interfering with HIV cell binding and infectivity.

In vitro enhancement of antibody responses by T delta cells expressing IgD receptors.

Previous in vivo studies have shown that murine T cells induced to express receptors specific for IgD (IgD-R) have humoral immunoaugmenting properties with respect to both primary and secondary antibody responses to various antigens. Such murine IgD-R+ T cells (T delta cells) belong to the CD4+ T cell population in contrast with human T delta cells which also include CD8+ cells. The purpose of these studies was to develop an in vitro assay system to examine the mechanism by which T delta cells facilitate enhanced antibody responses. Our studies demonstrate that B cell responses to both soluble and particulate antigens can, indeed, be enhanced in vitro by coculture with T cells previously induced to express IgD-R. This in vitro effect requires cognate interaction between T and B cells and is dependent on the presence of adherent cells or IL-1. T cell priming with antigen, while not a prerequisite, was found to result in more effective T delta-B cell interactions compared with naive T delta cells. Finally, evidence was obtained in support of an adhesion-mediated T delta-B cell interaction since the immunoaugmenting properties of T cells expressing IgD-R are completely blocked by the addition of very low doses of monomeric IgD to the cultures.

The immunoaugmenting properties of murine IgD reside in its C delta 1 and C delta 3 regions: potential role for IgD-associated glycans.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.

IgD-receptor-positive human T lymphocytes. II. Identification and partial characterization of human IgD-binding factor.

Membrane receptors specific for IgD (IgD-R) have been identified on murine CD4+ and human CD4+ and CD8+ T lymphocytes. Up-regulation of these IgD-specific receptors can be achieved by exposure of such T cells to various stimuli, including oligomeric or Ag cross-linked IgD, IL-2, IL-4, and T cell mitogens, such as PHA. Previous studies with murine IgD-R+ splenic T cells and IgD-R+ T hybridoma cells have demonstrated the existence of soluble IgD-binding factors (IgD-BF) that are shed or released into the medium in which these cells are grown. In our study, human peripheral blood T cells and IgD-R+ T hybridoma cells were examined for their ability to produce human IgD-BF. PHA stimulation of peripheral blood T cells results in their release of an IgD-specific factor with an apparent Mr of 70 kDa. IgD- Sepharose-purified IgD-BF was able to competitively inhibit rosetting of IgD-R+ T cells with IgD-coated RBC. Immunoblot assays in which alkaline phosphatase-conjugated human IgD myeloma protein was used as a probe, confirmed the IgD specificity of IgD-BF. An IgD-BF-specific mAb (LTB9) that also reacts with membrane IgD-R was produced after immunization of BALB/c mice with this factor. LTB9 was able to detect IgD-BF in the supernatants derived from human IgD-R+, tetanus toxoid-specific T hybridoma cells, H9-CEM1, and to stain membrane IgD-R by indirect immunofluorescence. Stimulation of H9-CEM1 cells with immobilized IgD resulted in up-regulation of membrane IgD-R expression, as measured cytofluorometrically with LTB9-stained cells, and potentiated release of IgD-BF from these cells. Finally, LTB9 as well as IgD-Sepharose, immunoprecipitated a 70-kDa protein from the lysates of biosynthetically labeled H9-CEM1 cells. Similar immunoprecipitation results were obtained with H9-CEM1-derived supernatants containing IgD-BF. Taken together, these results support the hypothesis that human T cell membrane IgD-R are released as soluble IgD-BF.

Specificity of the murine IgD receptor on T cells is for N-linked glycans on IgD molecules.

IgD receptors on murine T cells have been reported in this issue [Tamma, S. M. L., Amin, A. R., Finkelman, F. D., Chen, Y.-W., Thorbecke, G. J. & Coico, R. F. (1991) Proc. Natl. Acad. Sci. USA 88, 9233-9237] to bind either the first or third constant region of the heavy-chain of IgD molecules--findings that could not be satisfactorily explained by IgD amino acid sequences. We now find that boiled IgD molecules or low-Mr fragments from protease-digested IgD still inhibit binding of IgD-coated erythrocytes to IgD receptors. This inhibitory activity can be absorbed with the murine IgD-binding lectin from Griffonia simplicifolia 1 (GS-1) immobilized on Sepharose. N-linked glycans, obtained from N-glycanase-treated IgD and purified by binding to GS-1-Sepharose, also inhibit rosette formation of T-helper cells bearing receptors for IgD with IgD- or mutant IgD-coated erythrocytes. Deglycosylated IgD, produced by treatment with N-glycanase, no longer binds to the lectin and fails to inhibit IgD rosetting. Binding of intact IgD to T cells is also competitively inhibited by N-acetylgalactosamine, galactose, N-acetylglucosamine, and neoglycoproteins containing these sugars. These results clearly show that N-linked glycans, present in both the first and third constant regions of the delta heavy-chain domains, are prerequisites for binding of IgD to IgD receptors.

IgD receptors on murine T-helper cells bind to Fd and Fc regions of immunoglobulin D.

Receptors for immunoglobulins on animal cells invariably show specificity for Fc regions of the protein and are hence called Fc receptors. The present study shows that immunoglobulin D receptors present an exception to this rule. Binding of IgD-coated erythrocytes to murine IgD-receptor-bearing T-helper cells is competitively inhibited by IgD, by its Fab delta fragments, and by deletion mutants of IgD lacking (i) the first constant domain of the delta heavy chain (KWD1), (ii) that region plus the delta heavy-chain-hinge region (KWD6), or (iii) the third constant domain of the delta heavy chain (Gen. 24). KWD1, Gen. 24, or KWD6 mutants bind to T-helper cells bearing receptors for IgD independently of each other. Furthermore, Gen. 24 and KWD6 mutants also competitively inhibit binding of each other in cross-blocking experiments. These results show that the IgD receptors binds to the Fd delta and the Fc delta and cannot readily be explained by sequence homology between the two parts of the IgD molecule.

IgD-receptor-positive human T lymphocytes. I. Modulation of receptor expression by oligomeric IgD and lymphokines.

Studies with human myeloma-derived IgD have demonstrated the existence of IgD-R on peripheral blood T cells. These receptors, which are detected by rosetting with IgD-coated ox E (IgD-rosette-forming cells), are competitively inhibited by IgD, but not by IgM or IgG. Similar results were obtained with human T cell clones and T hybridomas derived from such clones either by rosetting assays or by staining with biotinylated-IgD. In agreement with studies of murine IgD-R+ cells, human IgD-R can be up-regulated by exposure of peripheral blood T cells, T cell clones, and hybridomas derived from such clones, to oligomeric IgD, but not monomeric IgD. Human IgD-R can also be induced by IL-2, IL-4, and IFN-gamma. In contrast with studies of murine IgD-R, which are expressed primarily by CD4+ cells, phenotyping studies show that both the CD4+ and CD8+ human T cell subsets are capable of expressing IgD-R.

Serum IgD levels in mice: effect of strain, age and autoimmune disease.

Serum IgD levels were studied in mice. Strain-related variability of serum IgD levels was noted, and advanced age was associated with markedly increased IgD levels in a large percentage of mice from all strains. Strains prone to spontaneously arising autoimmune disease had elevated IgD levels; in NZB mice this was already present very early after birth (one week), whereas in MRL mice the elevated serum IgD levels were first seen somewhat later (3 months). In contrast, mice with collagen type II (CII)-induced autoimmune arthritis had no increase in serum IgD. Injection of the immunomodulating agents LPS and complete Freund's adjuvant (CFA) did not have a significant effect on serum IgD levels, but IL-1 induced a significant decrease in IgD.

Release of IgD-binding factor by T cells under the influence of interleukin 2, interleukin 4, or cross-linked IgD.

Helper T cells with receptors specific for IgD have immunoaugmenting properties. We have now detected soluble IgD-binding factor in cell supernatants immobilized on nitrocellulose paper by their ability to bind 125I-labeled IgD. IgD-binding factor is released by normal splenic T cells stimulated with recombinant interleukin 2, recombinant interleukin 4, or crosslinked IgD in amounts paralleling the induction of IgD receptors on the cells. IgD receptors are constitutively produced by antigen-specific helper T-cell hybridomas 2H10 and A3.4C6. Incubation of these hybridoma cells with recombinant interleukin 2 increases release of IgD-binding factor while reducing expression of IgD receptors. Specificity of the binding factor for IgD is established by (i) competitive inhibition; (ii) the ability of the binding factor to bind radiolabeled IgD and not monoclonal IgE, IgG2a, or polyclonal IgG; and (iii) the removal of the binding factor on passage through an IgD-Sepharose column and recovery in a subsequent acid eluate.

Exposure to crosslinked IgD induces receptors for IgD on T cells in vivo and in vitro.

IgD is a surface immunoglobulin, which is coexpressed with IgM on greater than 90% of mature B cells, but its levels in serum are extremely low compared to those of IgM. It role as a surface receptor has been reemphasized by our recent findings that IgD receptors are induced on helper T cells by exposure to IgD and that such cells have immunoaugmenting properties. The present study shows that crosslinking of soluble IgD or of monomeric cell surface IgD is required and sufficient for the induction of T cells bearing receptors for IgD, both in vivo and in vitro. Effective IgD crosslinking in this respect can be obtained with antigen or with heterologous and immunogenic as well as nonimmunogenic allotype-specific anti-IgD. These results reinforce the concept that the induction of T cells bearing receptors for IgD is an integral component of the normal immune response.

Physiology of IgD. IX. Effect of IgD on immunoglobulin production in young and old mice.

Weekly i.p. injections of IgD from birth in (SJL X BALB/c)F1 mice were found to accelerate the development of IgG- and IgA-secreting cells and to increase the numbers of Ig-secreting cells of all isotypes in 17-28-day-old mice, but not in 7-10-day-old mice. Similarly, repeated weekly injections of IgD in normal adult BALB/c mice increased the numbers of reverse plaque-forming cells/spleen for all isotypes studied, including IgM, IgG1, IgG2, and IgA, but not for IgD itself. No such effect was observed in IgD-treated aged (20 months old) BALB/c mice. The absence of an effect of IgD on Ig secretion appeared to correlate with a lack of induction of receptors for IgD on T cells of the host, both in 7-10-day-old and in aged mice. In 7-10-day-old mice this lack of induction appeared due to their very low numbers of L3T4+ T cells. A comparison was made between the effect of a single injection of IgD or lipopolysaccharide (LPS) on numbers of Ig-secreting cells in the spleen determined 1-7 days after injection. Both agents caused increases, but the increase in IgM-producing cells was much greater after LPS (day 4), while IgD caused a relatively greater increase in IgG2 and IgA (days 4-7). Increases in IgG1 and IgG3-producing cells induced by LPS and IgD were of similar magnitude (days 6-7). IgD production, however, was not increased. The number of cells producing antibody of anti-trinitrophenyl (TNP) specificity was enhanced by LPS (day 4), but not by a single injection of IgD, although more than one injection of IgD caused a significant increase in anti-TNP-producing cells above background. LPS, but not IgD, caused B cell proliferation in vitro in the presence or absence of gamma-irradiated T delta cells. However, in vivo, IgD injections caused a significant increase in the percentage of lymphoid follicles with germinal centers in lymph nodes from 17-21-day-old and normal adult mice, but not in 7-10-day-old or aged mice. Such an effect was also absent in 24-28-day-old mice, where germinal center development, even in untreated mice, was very high.

Physiology of IgD. VIII. Age-related decline in the capacity to generate T cells with receptors for IgD and partial reversal of the defect with IL 2.

In previous studies we have shown that T cells from young mice can be induced to express receptors for IgD (T delta cells) after in vivo or in vitro exposure to IgD or IL 2. In the present study, mice of three different strains were used to study the effects of aging on the ability of splenic T cells to express such receptors. It is shown that T cells from mice older than 18 mo of age are deficient with respect to their ability to express receptors for IgD after in vivo or in vitro exposure to IgD. Similarly, IFN-gamma induces T delta cells in young but not in aged mice. In contrast, T delta cells can be induced in aged mice with IL 2, although to a lower percentage than in young mice. In agreement with our previously reported findings, the current study demonstrates the immunoaugmenting effect of IgD injections on the antibody response in young mice. This effect is absent in aged mice and thus correlates with the failure of IgD to induce T delta cells in such animals.

Physiology of IgD. VII. Induction of receptors for IgD on cloned T cells by IgD and interleukin 2.

The role of IgD in the immune response has remained elusive, although the predominance of IgD on the B cell surface and the paucity of IgD in serum have suggested a receptor function. In support of this hypothesis, it has recently been shown that receptors for IgD on helper T cells can be induced by exposure to IgD in vivo and in vitro. Such IgD receptor-positive T cells (i.e., T delta cells), detectable as RFC using IgD-coated SRBC, augment antibody responses. In this report, we demonstrate that cloned, antigen-specific T cells of helper phenotype show only very low percentages of IgD-RFC, if allowed to rest in vitro after antigen exposure in the absence of IL 2. Exposure to IgD or to IL 2 for 24 hr causes the IgD-specific RFC to increase as much as 25-fold to nearly 80%. Clones that have recently been stimulated with antigen, or T cell hybridomas prepared from such clones, exhibit 40 to 50% IgD-RFC before exposure and twofold higher levels after exposure to IgD. IL 2 also causes a dose-dependent induction of OgD-RFC in normal splenic T cells. Thus, antigen stimulation, IL 2 and IgD can all induce these receptors for IgD which presumably enable helper T cells to interact more effectively with IgD+ B cells.

Proliferative responses of T cells from SJL----F1 and F1----SJL bone marrow chimeras to SJL lymphoma cells.

RCS tumor cells induce marked proliferation of syngeneic SJL T cells in vivo and in vitro. Certain F1 hybrids of SJL mice give high proliferative responses to gamma-RCS, while other F1 hybrids give low responses. SJL----"non-responder" F1 and "non-responder" F1----SJL semiallogeneic bone marrow chimeras were prepared to study how the host environment affects the ability of T cells to give a proliferative response to gamma-RCS. The results indicate that T cells educated in an SJL host become responsive to RCS cells, while T cells educated in an (SJL X BALB/c)F1 host become unresponsive. This finding applies to both thymus and lymph node T cells. The unresponsiveness in F1 mice is not due to suppressor cells, since added F1 cells do not affect the proliferative response of SJL cells to gamma-RCS. Instead, it appears that RCS-specific T cells are either deleted in (SJL X BALB/c)F1 mice, or expanded in SJL mice as they develop. These findings are discussed in relation to the specificity of the responding T cells, for LPS activated syngeneic B cell blasts as well as RCS cells, and to the presence of a "leaky" thymus barrier in SJL mice for B cells.

Physiology of IgD. VI. Transfer of the immunoaugmenting effect of IgD with T delta-containing helper cell populations.

We show that the IgD-induced augmentation of the immune response to trinitrophenylated keyhole limpet hemocyanin can be transferred to syngeneic mice with spleen cells from IgD-injected donors. The augmenting activity is present in the Lyt-1+2-, L3T4+ T cell population and is absent from B cells. The ability of transferred T cells to augment the immune response correlates with the presence of a high frequency of Lyt-1+2- T cells that form rosettes with IgD-coated sheep erythrocytes (T delta cells). Such rosette-forming cells can also be induced by incubation of spleen cells from normal donors in IgD-coated petri dishes. Injection of normal spleen cells exposed to IgD-coated petri dishes together with antigen also augments the immune response of recipients. The existence of a regulatory circuit based upon interactions between T delta cells, antigen, B cell surface IgD, and serum IgD, is proposed.