A priori selection of the regularization parameters in emission tomography by Fourier synthesis.

This paper describes how the stability of the inverse problem underlying emission tomography can be measured and controlled in clinical settings. We show how the Lanczos approximation provides a way to regularize a certain class of iterative reconstruction algorithms through a given level of noise or resolution in the slices and for a given acquisition protocol. Moreover, we show how the same Lanczos approximation can be used to decide when the iterative reconstruction algorithm actually converges for a given machine precision. These ideas are illustrated by means of reconstructions of simulated and actual emission datasets.

Highly restricted spread of HIV-1 and multiply infected cells within splenic germinal centers.

The tremendous dynamics of HIV infection finds expression in the tempo of sequence diversification. Genetic diversity calculations require the clearance of a majority of infected cells, the obvious predator being anti-HIV immune responses. Indeed, infiltration of germinal centers (GCs) by HIV-specific CD8(+) cytotoxic T lymphocytes has been described. A corollary to this description would be limited diffusion of virus within lymphoid structures. HIV efficiently infects and replicates mainly in activated CD4(+) T lymphoblasts. These cells are found within GCs after their activation in the adjacent periarteriolar lymphoid sheath (PALS). Here GCs and PALS have been dissected from consecutive 10-micrometer sections through splenic tissue from three HIV-1-infected patients. Nested PCR amplification of the two first hypervariable regions of the env gene indicated that 38-78% of sections contained HIV-infected cells. Since there are several hundred CD4(+) T cells per GC section, approximately 0.09-0.64% harbor proviral DNA. Such a low frequency not only suggests that virions on the follicular dendritic cell surfaces do not readily infect adjacent T cells but also indicates highly restricted spread of HIV within GCs and the PALS. Sections were heavily infiltrated by CD8(+) cells, which, together with a large body of extant data, suggests that the majority of infected cells are destroyed by HIV-specific cytotoxic T lymphocytes before becoming productively infected. Finally, sequence analysis revealed that those HIV-positive cells were multiply infected, which helps explain widespread recombination despite a low overall frequency of infected cells.

The extracellular domain of CD4 regulates the initiation of T cell activation.

To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, confirming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb specific for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.

lck-independent inhibition of T cell antigen response by the HIV gp120.

Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 > or = 1 phase of the cell cycle.

Antigenic stimulation by BCG vaccine as an in vivo driving force for SIV replication and dissemination.

The impact of antigenic stimulation on the dynamics of simian immunodeficiency virus (SIV) replication was studied following repeated intravenous BCG inoculation of a SIV infected macaque. At the site of a delayed type hypersensitivity reaction to purified protein derivative of M. tuberculosis, a distinctive SIV variant was noted, probably as a result of the infiltration of activated antigen-specific T cell clones as opposed to infection by blood borne virus in situ. The dynamics of SIV quasispecies in peripheral blood suggests sequential waves of viral replication, illustrating the role of antigenic stimulation as a driving force in viral dissemination and pathogenesis.

Molecular analysis of the cytoplasmic domain of CD4: overlapping but noncompetitive requirement for lck association and down-regulation by Nef.

Expression of the HIV Nef protein results in the down-regulation of cell surface expression of CD4, with a di-leucine motif in the cytoplasmic domain of CD4 being required for this effect. However, our results indicate that this motif is not sufficient to confer sensitivity to down-regulation by Nef. Using site-directed mutagenesis and a transient expression system, we demonstrate that an alpha-helical stretch of amino acids of the cytoplasmic tail of CD4 is also required for the down-regulation of CD4 induced by Nef. Some CD4 mutations allowed the discrimination between PMA- and Nef-induced down-regulation, suggesting the existence of multiple pathways. In addition, our results demonstrate that this motif is involved in the association of CD4 with the tyrosine kinase p56lck, thus defining a multifunctional domain of CD4. Although there is overlap between the sequence requirement for lck association and susceptibility to Nef, we fail to detect any preferential decrease in lck association with CD4 when Nef is expressed during acute HIV infection. Altogether, these results demonstrate that there is an overlapping, but noncompetitive, sequence requirement in the cytoplasmic domain of CD4 for lck association and down-regulation by Nef.

Degradation of CD4 induced by human immunodeficiency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

The HIV-1-encoded Vpu protein induces a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum (ER). In this study, Vpu-induced degradation of CD4 in the ER was investigated by quantitative immunoprecipitation of CD4 following cotransfection of COS-7 cells with CD4 and Vpu expressors in the presence of brefeldin A, a drug that blocks protein transport from the ER to the Golgi complex. In order to precisely define the sequence(s) or structural element(s) in the CD4 cytoplasmic domain necessary for Vpu-induced degradation, a panel of deletion and substitution mutants in the cytoplasmic domain of CD4 was generated and analyzed. In agreement with previous reports, our deletion analysis indicates that a region encompassing amino acids 411 to 419 (KRLLSEKKT) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity. However, six specific substitution mutations within this region did not confer CD4 resistance to Vpu, suggesting that neither the amino acid sequence nor the charge of the amino acids in this region was critical to Vpu-induced CD4 degradation. A dileucine motif that is important for internalization of CD4 and Nef-induced CD4 down-regulation was also not required for Vpu-induced CD4 degradation. Interestingly, two substitution mutants (CD4EMKL and CD4MK407,11PP) located in a more proximal cytoplasmic region of CD4 abolished Vpu-induced CD4 degradation. Computer-assisted analysis of the substitution and deletion mutants conferring CD4 resistance to Vpu-induced degradation indicated that these mutations disrupted a putative alpha-helix formed in the proximal cytoplasmic region of CD4. Taken together, these studies strongly suggest that a structural element in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

LFA-1-mediated antigen-independent T cell adhesion is regulated by CD4 and p56lck tyrosine kinase.

We examined the role of CD4 and p56lck in the regulation of LFA-1-dependent T cell adhesion to B cells and to fibroblasts expressing ICAM-1 and HLA-DR by using various transfectant constructions. Although CD4 transfection in CD4low HUT78 T cell lines did not significantly modify their maximal binding to B cells and fibroblasts, it made the LFA-1-dependent adhesion sensitive to inhibition by anti-CD4 Ab, HIV-1 (env) gp 160, and a 12-mer peptide encompassing the 35-46 sequence of the beta 1 domain of the MHC class II molecule. CD4low HUT78 T cell adhesion to B cells was stable over 60 min, whereas expression of CD4 led to a transient adhesion. In addition, adhesion of CD4+ T cells to MHC class II- B cells was also stable. The CD4-dependent alteration of adhesion required the association of CD4 with p56lck because expression of mutant forms of CD4 unable to bind p56lck resulted in a lack of CD4-dependent regulation of adhesion. Herbimycin A, an inhibitor of tyrosine kinase activity, reversed the effect of CD4 transfection on adhesion. These results indicate that ligand binding to CD4 delivers a signal-inducing cell dissociation by activating p56lck tyrosine kinase. This regulatory pathway may provide a quick and reliable way for multiple and subsequent Ag-independent adhesion events of CD4+ T cells.

Association of p56lck with the cytoplasmic domain of CD4 modulates HIV-1 expression.

To investigate the role played by the cytoplasmic domain of the CD4 glycoprotein in the process of HIV infection, we have transfected two CD4-negative human T cell lines with cDNAs encoding the full-length CD4 and a truncated form of the molecule, lacking most of the cytoplasmic domain. Levels of viral replication were significantly higher in cells carrying the truncated version of CD4, in comparison with cells expressing the full-length CD4, as measured by the percentage of cells expressing viral p24 protein and the number of infectious particles released into culture supernatants. The extent of viral entry and reverse transcription was similar in each case, as monitored by an enzymatic test and quantitative PCR. Quantitative differences at RNA and protein levels were responsible for changes in viral production. To further characterize the mechanisms responsible for decreased rates of HIV replication in CD4-expressing cells we have treated the different cell lines, very early after HIV infection, with azidothymidine and soluble CD4, two antiviral agents that inhibit replication of HIV at different stages in the virus replicative cycle. Results from these experiments indicate that a cellular signal is mediated by the CD4 molecule, which negatively regulates the expression of viral DNA already present in such cells. This signal would be initiated following oligomerization of the CD4 molecule by the virus itself. Results from experiments with a CD4 construct containing mutations of the cysteine residues which are responsible for association of CD4 with p56lck demonstrate that p56lck is implicated in the transduction of the signal negatively regulating HIV replication.

Association of tyrosine kinase p56lck with CD4 inhibits the induction of growth through the alpha beta T-cell receptor.

The membrane glycoprotein CD4 enhances antigen-mediated activation of T cells restricted by class II molecules of the major histocompatibility complex (MHC). This positive function has been attributed to the protein tyrosine kinase p56lck (ref. 4), which is noncovalently associated with the cytoplasmic portion of CD4, and is activated on CD4 aggregation. Antigen presentation by MHC class II molecules coaggregates CD4 and the T-cell antigen receptor (TCR alpha beta-CD3). Thus, the mutual specificity of CD4 and TCR alpha beta for the MHC-antigen complex results in the juxtaposition of p56lck and TCR alpha beta-CD3. In contrast, anti-CD4 antibodies can abrogate antigen-induced, as well as anti-TCR-induced T-cell activation, indicating that CD4 might also transduce negative signals. The molecular basis for this opposing function remains unclear. Here we show that the CD4-p56lck complex prohibits the induction of activation signals through the TCR-CD3 complex when not specifically included in the signalling process. This negative effect does not require anti-CD4 treatment, indicating that the induction of distinct negative signals is probably not involved. Rather, the results demonstrate that the CD4-p56lck complex provides prerequisite signals for antigen-receptor-induced T-cell growth and thus characterize a molecular mechanism for functional constraints imposed on T-cell activation by the MHC.