David Leitenberg

Distinct Patterns of Membrane Microdomain Partitioning in Th1 and Th2 Cells

Here we show that activated Th1 and Th2 cells have distinct patterns of membrane compartmentalization into lipid rafts. TCR complex members are recruited efficiently to rafts and aggregate with rafts at the site of MHC/peptide contact in Th1 cells but not Th2 cells. TCR/raft association in Th1 cells is deficient in the absence of CD4, suggesting that CD4 aids recruitment of the TCR to rafts. We show differential utilization of rafts in Th1 and Th2 cells by cholesterol depletion studies, which alters calcium signaling in Th1 but not Th2 cells. Furthermore, Th2 cells have a decreased ability to respond to low-affinity peptide stimulation. These studies indicate that components of membrane microdomains are differentially regulated in functionally distinct CD4 T cells.

The Potency of TCR Signaling Differentially Regulates NFATc/p Activity and Early IL-4 Transcription in Naive CD4+ T Cells

The potency of TCR signaling can regulate the differentiation of naive CD4+ T cells into Th1 and Th2 subsets. In this work we demonstrate that TCR signaling by low-affinity, but not high-affinity, peptide ligands selectively induces IL-4 transcription within 48 h of priming naive CD4+ T cells. This early IL-4 transcription is STAT6 independent and occurs before an increase in GATA-3. Furthermore, the strength of the TCR signal differentially affects the balance of NFATp and NFATc DNA binding activity, thereby regulating IL-4 transcription. Low-potency TCR signals result in high levels of nuclear NFATc and low levels of NFATp, which are permissive for IL-4 transcription. These data provide a model for how the strength of TCR signaling can influence the generation of Th1 and Th2 cells.

Isoforms of the transmembrane tyrosine phosphatase CD45 differentially affect T cell recognition

Activation of T cells has been shown to require CD45. CD45 is expressed on T cells as distinct isoforms and these isoforms are expressed differentially on subsets of CD4 T cells. We have generated T cell lines expressing a T cell receptor (TCR) of known specificity, with or without CD4, and examined the effect of different CD45 isoforms on stimulation through the antigen receptor. We find that isoforms differ in their ability to participate in antigen recognition, with the null isoform that is predominantly found on memory CD4 T cells being the most effective. The ability of the CD4 T cells being the most effective. The ability of the CD45 ectodomain to differentially affect sensitivity to specific ligands represents a novel way of regulating the efficacy of signaling through a receptor without altering its specificity. It may play a crucial role both in immunological memory and during intrathymic maturation of T cells.

BIOCHEMICAL ASSOCIATION OF CD45 WITH THE T CELL RECEPTOR COMPLEX : REGULATION BY CD45 ISOFORM AND DURING T CELL ACTIVATION

CD45 is the predominant transmembrane tyrosine phosphatase in lymphocytes and is required for the efficient induction of T cell receptor signaling and activation. However, the regulation of CD45 activity and substrate specificity are poorly understood. In the present study, we demonstrate a basal biochemical association of CD45 with the T cell receptor complex that is regulated in part by CD45 isoform expression. Further, maintenance of CD45/TCR association is differentially regulated following TCR ligation with peptide: a partial agonist peptide induces CD45/TCR dissociation while an agonist peptide promotes sustained association in a CD4-dependent manner. These data suggest that T cell receptor signaling pathways may be modulated by altering access of CD45 to TCR-associated substrates involved in T cell activation.

Combinatorial Effect of T-Cell Receptor Ligation and CD45 Isoform Expression on the Signaling Contribution of the Small GTPases Ras and Rap1

ABSTRACT By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO+, but not CD45ABC+, T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO+ expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO+ T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC+ T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.

Epstein-Barr virus suspension cell assay using in situ hybridization and flow cytometry

We have developed a procedure for quantitative assay of Epstein-Barr virus (EBV)-infected cells in suspension in either latent or replicative phase using in situ hybridization and flow cytometry. The cells were hybridized with EBV-specific digoxigenin or biotin-labeled oligonucleotide probes, followed by binding to fluorescein-conjugated anti-digoxigenin or phycoerythrin-conjugated streptavidin, respectively. The cells hybridizing to the specific probes were quantitated by flow cytometry. A strong shift in fluorescence intensity (20-fold) was observed when the EBV-positive culture cells were hybridized with a specific EBER1 antisense probe. The sensitivity of the assay was at least one positive cell out of 9,000 beyond the normal control mean +/- 2 S.D. We performed two-color in situ hybridization/flow cytometry using probes to an EBV replication phase-specific mRNA and EBER1 on B95-8 cells in which a small portion (2-4%) of cells induce spontaneously into the replicative phase. In addition, we have developed a method for simultaneous analysis of the cell surface phenotype and EBV nucleic acid content in individual cells.