Signalling Capacity of TCR Antagonists

The earliest documented events following engagement of TCRs by their specific ligands, peptide/MHC complexes, involves the activity of Src and Syk/ZAP-70 family protein tyrosine kinases and the phosphorylation of a number of their cellular substrates. These include the immunoreceptor, tyrosine-based activation motifs (ITAM) of the TCRx and CD3 proteins, the adaptor proteins LAT and SLP-76, and the proto-oncogene product, Vav, as well as proteins of less-defined function, such as SLAP-130/FYB. Some of these activation-induced tyrosine phosphorylation events are exquisitely sensitive to the affinity of interaction between the TCR and its MHC/peptide ligand. Studies on single amino acid-substituted antigenic peptide variants, so-called altered peptide ligands (APL) have indicated that low-affinity interactions often lead to an incomplete pattern of tyrosine phosphorylation. Initial studies indicated that stimulation by APL led to a preponderance of an incompletely phosphorylated z chain, with the resulting accumulation of a low molecular weight mass TCRz and the failure to phosphorylate and activate ZAP-70. In contrast to these signalling defects, we have demonstrated that antagonist peptides are capable of inducing the signalling pathway that is required for cytoskeletal reorganization in T cells, the formation of stable T cell/APC conjugates, and the localization of certain T cell proteins to the areas of contact between APC and T cell. This TCR antagonist induced signalling pathway is characterized by the phosphorylation and activation of Vav guanine nucleotide exchange factor activity and subsequent activation of the small G protein, Rac.

We have recently investigated the role played by the protein tyrosine kinases Fyn, Lck, and ZAP-70 in antagonist-induced signalling. Antagonist stimulation increased tyrosine phosphorylation and kinase activity of Fyn severalfold, whereas little or no increase in Lck and ZAP-70 activity was observed. Second, TCR stimulation of Lck-, Fyn+ Jurkat cells induced strong tyrosine phosphorylation of Vav. In contrast, minimal increase in tyrosine phosphorylation of Vav was observed in Lck+, Fyn- Jurkat cells. Finally, study of T cells from Fyn-deficient TCR transgenic mice also showed that Fyn was involved in the tyrosine phosphorylation and activation of Vav induced by both antagonist and agonist peptides. The deficiency in Vav phosphorylation in Fyn-deficient T cells was associated with a defect in the formation of APC-T cell conjugates when T cells were stimulated with either agonist or antagonist peptide. In summary, we conclude that Vav is a selective substrate for Fyn, especially under conditions of low-affinity TCR-mediated signalling, and the signalling pathway involving Fyn, Vav, and Rac-1 is required for the cytoskeletal reorganization that leads to T cell-APC conjugates and the formation of the immunologic synapse.

Activation of naïve CD4 T cells by anti-CD3 reveals an important role of Fyn in Lck-mediated signaling

Signaling through the T cell antigen receptor is initiated by the activity of the Src family tyrosine kinases, Fyn and Lck. It has been shown in numerous studies using Lck deficient cell lines and mice whose peripheral T cells are deficient in Lck that this enzyme plays a critical upstream role in the signaling cascade leading to T cell activation, expansion and differentiation. In contrast to Lck, the role of Fyn kinase in T cell activation and development is less well defined. Fyn deficiency has little or no effect on T cell development in the thymus and peripheral T cells from Fyn deficient mice, to the extent they have been studied, show variable and incomplete defects in mounting immune responses. These findings are consistent with the concept that Fyn is, for the most part, a redundant Src kinase without a unique function in the signaling to T cells. However, over the past several years a number of studies have shown that Fyn has its own unique substrates, some of which play significant roles in T cell activation. These include ADAP (SLAP-130/FYB), Pyk2, WASp and CBP. Other indications that Fyn plays some unique role in TCR mediated signaling come from our previous studies that have shown that stimulation of T cells with low affinity ligands that function as TCR antagonists leads to preferential activation of Fyn in the absence of any detectable changes in the activity of Lck or ZAP-70.

Because of these more recent findings, we have reinvestigated the capacity of Fyn deficient T cells to respond to TCR mediated signaling. For this purpose, we compared naïve CD4 T cells from Fyn deficient and wild-type mice that contained a TCR transgene for their capacity to respond to either antigen presented by APCs or to respond to antibody mediated CD3 crosslinking. Our results demonstrate that the capacity for Fyn-deficient T cells to be activated is critically dependent on the mode of stimulation and the prior experience of the T cells. Naïve Fyn-/- CD4 T cells stimulated by CD3 crosslinking had profound defects in the early signaling events of tyrosine phyosphorylation and calcium response which resulted in a markedly reduced ability to produce IL-2 and to proliferate. In contrast, previously activated Fyn-/- T cells showed no such activation defects, nor did Fyn-/- naïve T cells stimulated with antigen pulsed APCs.

Surprisingly the signaling defect following CD3 crosslinking of Fyn-/- T cells included the Lck mediated activation of ZAP-70 and the subsequent phosphorylation of LAT by ZAP-70. Thus, stimulation restricted to TCR crosslinking requires Fyn to either activate Lck or induce the translocation of Lck into the region of the plasma membrane where TCR and ZAP-70 are located. In the presence of T cell-APC interaction and the formation of an immunologic synapse or when CD3 and CD4 are co-crosslinked with antibodies the Fyn requirement is no longer observed. Although the physiologic significance of these results is unclear these data together with our previous studies that Fyn activation preferentially occurs following stimulation with low affinity ligands suggest the possibility that TCR interactions with low affinity self ligands on dendritic cells stimulate sufficient Fyn activity to activate a small amount of Lck/ZAP-70 and, in the absence of an immunologic synapse, thus generates the signals required for survival and homeostatic proliferation.

Katsuji Sugie, Ph.D., Research Scientist

Introduction

Allergy is caused by unregulated activation of Th2-type CD4 T cells and formation of allergen-specific IgE antibodies. Our project was initiated by Dr. Kimishige Ishizaka, who discovered IgE in the late 1960's and an IgE-regulating cytokine glycosylation-inhibiting factor(GIF) in 1980's. GIF is a 13-kDa polypeptide. Although the gene encoding GIF has been identified, the role of this molecule in the immune regulation remains unclear. We have documented that GIF secreted from T cells binds to B cells and activated T cells. Our in vitro experiments indicated that this cytokine inhibits B cell secretion of IgE and IgG1. GIF also seems to regulate antigen-presenting function of B cells since it inhibited B cell antigen receptor(BCR)-mediated antigen uptake. We recently found that T-dependent antibody formation is enhanced in GIF-/- mice and that GIF-/- CD4 cells differentiate toward a Th2 phenotype. These data suggest that GIF is a unique cytokine that regulates CD4 T cell-dependent immune responses, although the mechanism seems complex. Our goal is to clarify the molecular mechanism of this regulation.

Structure and posttranslational modification of GIF

GIF is encoded by the same gene as macrophage migration inhibitory factor(MIF). There is a single functional gene for GIF/MIF in the mouse genome. GIF/MIF mRNA as well as protein is expressed in all kinds of eucaryotic cells so far tested. The nucleotide sequence shows no homology with other proteins. GIF monomers are connected to each other by a hydrophobic interaction to form a trimer.

GIF was originally discovered in T cell culture supernatants by the activity of regulating IgE synthesis. Injection of T cell-secreted GIF into mice inhibited the IgE and IgG1 antibody responses to DNP-Ova. Recently a mass spectrometric analysis revealed that GIF secreted from T hybridoma cells has undergone posttranslational modifications: cysteinylation of C60 and phosphorylation of S91. The cysteinylation was found to be required and sufficient for this cytokine to bind to target cells. It remains to be clarified what type of cells secretes cysteinylated GIF and what enzyme catalyzes this modification.

Bioactivity of GIF/MIF

In contrast to GIF secreted from T cells, unmodified wildtype recombinant GIF/MIF not only fails to bind to cells but also has no reproducible effect on cells. However, chemical modifications including cysteinylation of recombinant GIF at C60 render this molecule capable of binding to target cells and inhibiting IgE and IgG1 antibody responses to DNP-Ova in vivo. In vitro experiments demonstrated that the C60 modification is required for GIF to inhibit B cell IgE secretion induced by LPS and IL-4 and B cell antigen uptake through BCR.

The availability of GIF(MIF)-deficient mice has made it possible to assess the biological relevance of this molecule. GIF(MIF)-/- mice were reported to be more susceptible to Leishmania major infection, which suggests that this molecule is involved in regulating Th differentiation. It was also reported that GIF(MIF)-/-mice failed to develop experimental colitis, which implicates GIF in the activation of macrophages in this model disease. We have begun to compare GIF+/+ and -/- mice for antibody responses to various immunogens. Our preliminary data suggest that T-dependent antibody formation was enhanced in GIF-/- mice. GIF-/- mice also showed a reduced generation of Th1 effector cells. To determine what cells produce GIF and what cells respond to this molecule, we have generated OTII and DO11.10 TCR transgenic mice on GIF+/+ and -/- genetic backgrounds. The role of GIF derived from T cells and non-T cells will be examined by stimulating these antigen-specific T cells in vitro and in vivo by antigen and GIF+/+ or -/- APCs.

Katusji Sugie, Ph.D.

GIF receptor

To define the relevance of GIF, it is of particular importance to clarify the structure of GIF receptor. To perform a receptor binding assay, GIF was purified from T cell culture supernatant and labeled with 125I. Using various derivatives of recombinant GIF, we have documented that unmodified wildtype GIF does not bind to the receptor and that C60-modification enables the molecule to bind to the receptor. We have recently developed a receptor binding assay using biotinylated GIF in flow cytometry. This new assay was useful in confirming that GIF needs to be modified to bind to the receptor. We performed multiple-color flow cytometry to identify the target cells for this cytokine, which include B cells and activated CD4 and CD8 T cells. It was recently published that MIF, which is identical to unmodified recombinant GIF, binds to the cell surface moiety of invariant chain of class II MHC molecule(Ii). We have obtained Ii-/- mice and found that GIF binds to cells regardless of Ii expression, which indicates that GIF receptor is not Ii. We have biochemically identified GIF receptor on the surface of target cells by binding GIF covalently to target cells using chemical crosslinkers. Work is in progress to determine the structure of the receptor.

Our work is supported by NIH grants R21 AI47948 and R01 AI56211.