SummaryHuman immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and synrytium formation . Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain ofCD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and synrytium formation by a number of HIV isolates are blocked . These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion .C D4 is the high affinity cellular receptor for HIV and is found predominantly on thymus-derived T lymphocytes (1) . The structure predicted for CD4 is an extracellular region of four tandem domains having homology with members of the Ig superfamily, followed by a transmembrane domain and a cytoplasmic region (2, 3) . HIV binds CD4 via the major envelope glycoprotein, gp120, and recent genetic studies have localized the high affinity gp120 binding site on CD4 to -12 amino acids within the NH2-terminal, Ig-like (V1) domain (4-6). Similarly, residues have been defined in the COOH-terminal half of gp120 that are critical for CD4 binding (7,8). It is clear that the interaction between CD4+ human cells and cells expressing HIV envelope is sufficient to induce synrytium formation (9-11), and that virus entry is by pH-independent fusion ofthe virus and cell membranes (12, 13) . Little else is known, however, about the events subsequent to binding that lead to membrane fusion .Recently, evidence from two studies suggests that CD4 may play a role in cell-cell fusion (synrytium formation) additional to envelope binding . In the first study, single residue substitutions made in the CDR-3 analogous loop of the V1 domain of CD4 resulted in mutant molecules that were unable to facilitate synrytium formation, even though they mediated both virus binding and infectivity to an extent comparable with wild-type CD4 (14). In the second, chemical mutagenesis of a CD4+ T cell line resulted in CD4 molecules that bound gp120 but mediated synrytium formation poorly (15). Although the mechanism of inhibition of cell fusion was not elucidated in either study, the findings indicate that CD4 participates in events subsequent to gp120 binding that are required for membrane fusion. mAbs reactive with CD4/V1 are highly efficient at inhibiting the binding of gp120 to CD4, and thereby prevent infection and synrytium formation (6,(16)(17)(18)(19) . In contrast, a mAb reactive with the third or fourth CD4 domains has been shown not to inhibit gp120 binding (18,19) . To investigate whether regions of CD4 oth...

CD4' T cell lines were generated from the spleens of diabetic NOD mice against crude membrane preparations derived from a rat insulinoma. Adoptive transfer of these lines into neonatal mice confirms that overt diabetes is induced by v-IFN-secreting Thl cells, whereas transfer of IL-4-secreting Th2 cells resulted in a nondestructive peni-islet insulitis. Analysis of the antigens recognized by individual T cell clones from the Thl line included reactivity against an insulinoma membrane fraction enriched in proteins of -38 kD. Immune responses to the same antigen preparation have been associated with T cell clones derived from human insulin-dependent diabetes mellitus. The specificity of Th2 cells includes reactivity to a fraction enriched in proteins of 30 kD. The data suggest that in insulin-dependent diabetes mellitus the balance between fB cell destruction, associated with intra-islet infiltration, and nondestructive (potential protective) peri-islet insulitis may depend on both the antigens recognized, and the prevailing cytokine environment. (J. CliA. Invest 1995. 95:2979-2985

Immunogenicity, manufacturing feasibility, and safety of a novel, autologous dendritic cell (DC)-based immunotherapy (AGS-004) was evaluated in ten human immunodeficiency virus type 1 (HIV-1)-infected adults successfully treated with antiretroviral therapy (ART). Personalized AGS-004 was produced from autologous monocyte-derived DCs electroporated with RNA encoding CD40L and HIV antigens (Gag, Nef, Rev, Vpr) derived from each subjects's pre-ART plasma. Patients received monthly injections of AGS-004 in combination with ART. AGS-004 was produced within a mean of 6 weeks and yielded 4-12 doses/subject Full or partial HIV-specific proliferative immune responses occurred in 7 of 9 evaluable subjects. Responses were specific for the AGS-004 presented HIV antigens and preferentially targeted CD8 + cells. Mild adverse events included flulike symptoms, fatigue, and injection site reactions. No evidence of autoimmunity, changes in viral load, or significant changes in absolute CD4 + and CD8 + T cell counts were observed. This pilot study supports the further clinical investigation of AGS-004.

hsCD83 alone was capable of inducing kidney allograft tolerance through a mechanism involving Tol-DC generation and, at least in part, indoleamine 2,3-dioxygenase activity. Because sCD83 is of human origin, the therapeutic approach used in our mouse transplant model holds significant promise for clinical transplantation.

Bone marrow-derived dendritic cells (DCs) are cells of the immune system that have been used as a tool to boost, modulate, or dampen immune responses. In the context of autoimmunity, DCs can be modified to express immunoregulatory products encoded by transgenes, and used therapeutically in adoptive cellular therapy. DCs that were lentivirally transduced (lt) to express interleukin 4 (IL-4) can significantly delay or prevent the onset of autoimmune diabetes in nonobese diabetic (NOD) mice. However, modifying cells using viral vectors carries the dual risk of oncogenicity or immunogenicity. This study demonstrates that NOD DCs, electroporated with "translationally enhanced" IL-4 mRNA (eDC/IL-4), can be equally efficient therapeutically, despite the reduced amount and shorter duration of IL-4 secretion. Moreover, a single injection of eDC/IL-4 in NOD mice shortly after the onset of hyperglycemia was able to maintain stable glycemia for up to several months in a significant fraction of treated mice. Treatment with eDC/IL-4 boosted regulatory T (Tregs) cell functions and modulated T helper responses to reduce pathogenicity. Thus, treatment with DCs, electroporated with modified IL-4 mRNA to express IL-4 for up to 24 hours, constitutes a viable cellular therapy approach for the regulation of autoimmune diabetes, as a preferred alternative to the use of viral vectors.