Background: In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation.Methods: We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture.Results: Fused cells are detected as double‐fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV‐glycoprotein–related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti‐CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4‐expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion).Conclusions: The method's simple technical and minimal cell‐invasive procedures, as well as its non‐ambiguous automatic numerical quantification should be useful for the study of factors influencing cell‐cell fusion. Cytometry 47:100–106, 2002.

Interactionin vitrobetween cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006,J. Virol. Methods138, 17–23; López-Balderas et al., 2007,Virus Res.123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue.

T CD4+ cells are central to the adaptive immune response against pathogens. Their activation is induced by the engagement of the T-cell receptor by antigens, and of co-stimulatory receptors by molecules also expressed on antigen presenting cells. Then, a complex network of intracellular events reinforce, diversify and regulate the initial signals, including dynamic metabolic processes that strongly influence both the activation state and the differentiation to effector cell phenotypes. The regulation of cell metabolism is controlled by the nutrient sensor adenosine monophosphate-activated protein kinase (AMPK), which drives the balance between oxidative phosphorylation (OXPHOS) and glycolysis. Herein, we put forward a 51-node continuous mathematical model that describes the temporal evolution of the early events of activation, integrating a circuit of metabolic regulation into the main routes of signaling. The model simulates the induction of anergy due to defective co-stimulation, the CTLA-4 checkpoint blockade, and the differentiation to effector phenotypes induced by external cytokines. It also describes the adjustment of the OXPHOS-glycolysis equilibrium by the action of AMPK as the effector function of the T cell develops. The development of a transient phase of increased OXPHOS before induction of a sustained glycolytic phase during differentiation to the Th1, Th2 and Th17 phenotypes is shown. In contrast, during Treg differentiation, glycolysis is subsequently reduced as cell metabolism is predominantly polarized towards OXPHOS. These observations are in agreement with experimental data suggesting that OXPHOS produces an ATP reservoir before glycolysis boosts the production of metabolites needed for protein synthesis, cell function, and growth.

The adaptive immune response is initiated by the interaction of the T cell antigen receptor/CD3 complex (TCR) with a cognate peptide bound to a MHC molecule. This interaction, along with the activity of co-stimulatory molecules and cytokines in the microenvironment, enables cells to proliferate and produce soluble factors that stimulate other branches of the immune response for inactivation of infectious agents. The intracellular activation signals are reinforced, amplified and diversified by a complex network of biochemical interactions, and includes the activity of molecules that modulate the activation process and stimulate the metabolic changes necessary for fulfilling the cell energy demands. We present an approach to the analysis of the main early signaling events of T cell activation by proposing a concise 46-node hybrid Boolean model of the main steps of TCR and CD28 downstream signaling, encompassing the activity of the anergy factor Ndrg1, modulation of activation by CTLA-4, and the activity of the nutrient sensor AMPK as intrinsic players of the activation process. The model generates stable states that reflect the overcoming of activation signals and induction of anergy by the expression of Ndrg1 in the absence of co-stimulation. The model also includes the induction of CTLA-4 upon activation and its competition with CD28 for binding to the co-stimulatory CD80/86 molecules, leading to stable states that reflect the activation arrest. Furthermore, the model integrates the activity of AMPK to the general pathways driving differentiation to functional cell subsets (Th1, Th2, Th17, and Treg). Thus, the network topology incorporates basic mechanism associated to activation, regulation and induction of effector cell phenotypes. The model puts forth a conceptual framework for the integration of functionally relevant processes in the analysis of the T CD4 cell function.

Upon antigen stimulation and co-stimulation, CD4⁺ T lymphocytes produce soluble factors that promote the activity of other immune cells against pathogens or modified tissues; this task must be performed in presence of a variety of environmental cytokines, nutrient, and oxygen conditions, which necessarily impact T cell function. The complexity of the early intracellular processes taking place upon lymphocyte stimulation is addressed by means of a mathematical model based on a network that integrates variable microenvironmental conditions with intracellular activating, regulatory, and metabolic signals. Besides the phenotype subsets considered in previous works (Th1, Th2, Th17, and Treg) the model includes the main early events in differentiation to the T_FH phenotype. The model describes how cytokines, nutrients and oxygen availability regulate the differentiation of naïve CD4⁺ T cells into distinct subsets. Particularly, it shows that elevated amounts of an all-type mixture of effector cytokines under optimal nutrient and oxygen availability conduces the system towards a highly-polarized Th1 or Th2 state, while reduced cytokine levels allow the expression of the Th17, Treg or T_FH subsets, or even hybrid phenotypes. On the other hand, optimal levels of an all-type cytokine mixture in combination with glutamine or tryptophan restriction implies a shift from Th1 to Th2 expression, while decreased levels of the Th2-inducing cytokine IL-4 leads to the rupture of the Th1-Th2 axis, allowing the manifestation of different (or hybrid) subsets. Modeling proposes that, even under reduced levels of pro-inflammatory cytokines, the sole action of hypoxia boost Th17 expression.