Immunoglobulin-like transcript 3 (ILT3) and ILT4 belong to a family of inhibitory receptors expressed by human monocytes and dendritic cells. We show here that CD8+CD28(-) alloantigen-specific T suppressor (TS) cells induce the up-regulation of ILT3 and ILT4 on monocytes and dendritic cells, rendering these antigen-presenting cells (APCs) tolerogenic. Tolerogenic APCs show reduced expression of costimulatory molecules and induce antigen-specific unresponsiveness in CD4+ T helper cells. Studies of human heart transplant recipients showed that rejection-free patients have circulating TS cells, which induce the up-regulation of ILT3 and ILT4 in donor APCs. These findings demonstrate an important mechanism of immune regulation.

CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-alpha (TNF-alpha) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-alpha receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.

SummaryCD4 + T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed T helper function. The surface structures on activated CD4 + T cells that mediate this function are not fully known. We previously reported the isolation of a functionally unique subclone of the Jurkat leukemic T cell line (D1.1) that constitutively expressed contact-dependent helper effector function. To identify T cell surface molecules that mediate contact-dependent T helper function, a monoclonal antibody (mAb), designated 5c8, was generated that inhibits Dl.l-mediated B cell activation and immunoprecipitates a novel 30-kD protein structure from surface-iodinated D1.1 cells. Normal CD4 + T cells express 5c8 antigen (Ag) transiently after activation by phorbol myristate acetate and phytohemagglutinin with maximal expression 5-6 h after activation and absence of expression by 24 h. In contrast, neither resting nor activated CD8 § T cells express 5c8 Ag. In functional studies, mAb 5c8 inhibits the ability of fixed, activated CD4 § T cells to induce B cell surface CD23 expression. In addition, mAb 5c8 inhibits the ability of CD4 + T cells to direct terminal B cell differentiation driven by pokeweed mitogen. Taken together, these data suggest that 5c8 Ag is a novel, activation-induced surface T cell protein that is involved in mediating a contactdependent element of the helper effector function of CD4 § T lymphocytes.I n a contact-dependent process, termed T helper (Th) function, CD4 + T lymphocytes direct the activation and differentiation of B lymphocytes and thereby regulate the humoral immune response by modulating the specificity, secretion and isotype-encoded effector functions of antibody molecules (1-7). The T cell surface molecules that mediate the contact-dependent elements of Th cell function are not fully known.The process by which T cells help B cells to differentiate has been divided into two distinct phases: the inductive and the effector (8, 9). In the inductive phase, resting T cells contact antigen-primed B cells and this association allows clonotypic TCR-CD4 complexes to interact with Ia/Ag complexes on B cells (5, 10-16). TCP,/CD4 recognition of Ia/Ag results in the formation of stable T-B cognate pairs and bidirectional T and B cell activation (17-22). In the effector phase, activated T cells drive B cell differentiation by secreting lymphokines (23, 24) and by contact-dependent stimuli (20,(25)(26)(27)(28)(29)(30)(31)(32), both of which are required for T cells to drive small, resting B cells to terminally differentiate into Ig-secreting cells (25, 33, 34).Although the inductive phase of T cell help is Ag dependent and MHC restricted (5, 10-15, 34, 35), the effector phase of Th function can be Ag independent and MHC nonrestricted (25, 28, 30, 34,(36)(37)(38)(39)(40)(41)(42)(43)). An additional contrasting feature is that the inductive phase of T cell help often requires CD4 molecules and is inhibited by anti-CD4 mAb (16), whereas help...

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF-gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation.

Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10–30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.