Minimal tumor necrosis factor receptor binding protein: optimum biological activity of a truncated p55 soluble tumor necrosis factor receptor-IgG fusion protein.

We have previously demonstrated, using expressed deletion constructs, that the fourth membrane proximal cysteine-rich repeat of the p55 TNF receptor (TNF-R) is not required for binding of tumour necrosis factor-alpha (TNF) or lymphotoxin-alpha (LT; tumour necrosis factor-beta). We and others have also shown that the soluble p55 TNF-R, rendered dimeric by fusion to an IgG backbone is extremely effective at neutralizing the harmful effects of TNF overproduction, such as in toxic shock. Here we address the question of how the TNF binding properties of the truncated TNF-R comprising the three distal cysteine-rich repeats (delta4 TNF-R), when fused with an IgG backbone, compare with those of the full length soluble receptor. We constructed several versions of the soluble delta4 TNF-R, on a complete IgG heavy chain backbone and on an IgG lacking the CH1 (first constant region) domain. The constructs were expressed with an Ig or native TNF receptor leader sequence and altered or native N terminal sequence, to compare efficiency of expression. When compared with a full length, soluble receptor Ig fusion protein, the affinity of all for TNF was identical, as were their activities in in vitro binding and cytotoxicity assays. In vivo studies showed that the delta4 and wild type fusion proteins afforded equivalent protection against LPS-induced lethality. However, the delta4 proteins exhibited a significantly lower affinity for LT, and reduced activity in LT binding and cytotoxicity assays. We conclude that the truncated TNF receptor IgG fusion protein is as effective at neutralizing TNF activity as the full length soluble receptor fusion protein. Its lower affinity for LT may make it a more selective agent in blocking the action of TNF, while causing less interference with the action of LT. Also its smaller size may make it a more useful therapeutic agent as it may be less immunogenic than the full length receptor.

Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor.

To generate the full diversity of antibody heavy-chain genes, hundreds of dispersed germline V segments must undergo recombination following D-J segment joining. Here we report that this process is regulated by the alpha-chain of the receptor for interleukin-7, a cytokine that stimulates B-cell lymphopoiesis. D-J joining occurs normally in immature B lymphocytes from mice lacking the alpha-chain of the interleukin-7 receptor (IL-7Ralpha). But recombination of V segments is progressively impaired as their distance increases upstream of D/J, causing infrequent rearrangement of most V segments, which markedly reduces diversity. This is not simply due to defective cell proliferation or impaired recombinase expression. Rather, germline transcripts from distal, unrearranged V segments, a marker of chromatin changes that precede recombination, are specifically silenced. So too is expression of Pax-5, which binds to heavy-chain locus control elements and normally stimulates recombination, suggesting a mechanism for these effects. Thus ligands of the interleukin-7 receptor deliver an extrinsic signal that targets V segment recombination in the heavy-chain locus by altering the accessibility of DNA substrates to the recombinase. This mechanism augments the recombinational diversity of the primary antibody repertoire.

The interleukin-7 receptor alpha chain transmits distinct signals for proliferation and differentiation during B lymphopoiesis.

The interleukin 7 receptor (IL7R), which contains a unique alpha chain and a gamma chain shared by other cytokine receptors, is indispensable for normal lymphocyte development. The basis for this role is poorly understood. Here we show that the IL7R alpha chain not only causes progenitors to proliferate, but also has a distinct activity in inducing differentiation. First, we identify a single cytoplasmic tyrosine residue in the IL7R alpha chain that is essential for cell cycle entry and proliferation dependent on phosphatidylinositol 3-kinase. We use a mutant alpha chain in which this residue has been altered to reconstitute B lymphopoiesis by retrovirus-mediated gene transfer in cultures of bone marrow from mice deficient in IL7R alpha chain. The mutation abrogates the proliferation of B-lymphocyte progenitors, but reveals a novel function of the alpha chain in promoting immunoglobulin heavy chain gene rearrangement leading to B-cell differentiation. This function is lost (but proliferation sustained) when the cytoplasmic domain of IL7R alpha is replaced by corresponding sequences from the IL2R, despite the similarity on their signalling mechanisms. Thus, the signals which mediate a differentiative function of the IL7R in B lymphopoiesis are specific and distinct from those causing proliferation.

Characterization of ligand binding by the human p55 tumour-necrosis-factor receptor. Involvement of individual cysteine-rich repeats.

Two soluble tumour-necrosis-factor-alpha(TNF)-binding proteins are derived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF-mediated inflammatory processes, including diseases such as rheumatoid arthritis, by competing with the cell surface receptors for TNF and lymphotoxin (LT, tumour-necrosis factor beta). The extracellular domains of the two receptors each contain four similar cysteine-rich repeats of about 40 amino acids, in common with several other cell surface proteins including the p75 nerve-growth-factor receptor and the CD40 and Fas antigens. The aim of this study was to characterize the involvement of the four cysteine-rich repeats of the human p55 TNF receptor in TNF and LT binding by both membrane-bound and soluble forms of the receptor. Individual repeats were systematically deleted by PCR mutagenesis and the variants transiently expressed in COS cells. Immunoprecipitated receptor variants exhibited the expected sizes on SDS/PAGE gels, and bound a panel of conformation-dependent anti-(TNF receptor) antibodies. Binding of TNF by the four soluble derivatives was compared with binding by the wild-type soluble receptor using a TNF-affinity column and a BIAcore Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and 125I-TNF binding to U937 cells. delta 4, which lacks the fourth cysteine-rich repeat, bound TNF comparably with the full-length soluble receptor. TNF-binding affinity was unaltered by deletion of the fourth membrane-proximal cysteine-rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine-rich repeat is not required for TNF binding. In contrast, both the soluble and the transmembrane derivatives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that this may be due to indirect conformational change, rather than the removal of essential epitopes, our results suggest that the first three repeats are each required for TNF binding by both the soluble and the cell-surface receptor.

Autoantigen recognition by thyroid-infiltrating T cells in Graves disease.

Graves disease is a common form of human autoimmune thyroiditis. It shares many pathological features and HLA associations with other, less easily studied, organ-specific autoimmune conditions such as insulin-dependent diabetes mellitus, and hence it is also a useful model for understanding these other diseases. We have previously shown that thyroid-infiltrating T cells in Graves disease that have been recently activated in vivo specifically recognize autologous thyroid epithelial cells. However, the autoantigens involved were not defined. In this study, we have made use of antigen-independent T-cell cloning techniques to show that at least three different thyroid antigens, three different epitopes on a single antigen, and two HLA class II elements are involved in this recognition process in a single individual. This demonstrates that T cells that are present and activated at the site of a human autoimmune disease may show considerable heterogeneity in their recognition of autoantigen on the target tissue. This contrasts with the limited heterogeneity recently reported in some animal models and has potentially important implications for both our understanding of the autoimmune process in humans and the design of immunotherapies to reverse it.