Although TNF has the potential to serve as a potent antitumor treatment, its use is currently limited by high systemic toxicity and rapid clearance from the circulation. To address these issues, Krippner-Heidenreich et al. (p. 8176 ) developed a single-chain TNF derivative (scTNF) consisting of the three monomers of human TNF connected by dipeptide linkers. The development of scTNF circumvented the difficulty of creating bulky trimers in TNF fusion protein approaches to immunotherapy and resulted in a protein with enhanced binding affinity compared with wild-type TNF. scTNF had similar activity as wild-type TNF in the activation of NF-κB and JNK and in cytotoxicity assays. Introduction of the dipeptide linkers during scTNF creation did not induce formation of higher aggregates, nor did it affect TNFR-specific interaction sites on the molecule. scTNF was found to be much more stable than wild-type TNF both in vitro and in vivo, yet had much lower liver toxicity than wild-type TNF. Finally, the authors compared the antitumoral activity of scTNF vs wild-type TNF and found that their new reagent was as effective as wild type or more effective, depending on the tumor model that was used. The development of scTNF thus introduces a new strategy for TNF-based therapeutics.

A loss of memory T cell responses is known to be associated with HIV disease progression. Ladell et al. (p. 7907 ) studied patients who ingested deuterated water to analyze the in vivo lifespan and phenotype of long-lived memory CD8+ T cells in HIV-infected and uninfected individuals. Analysis of CD8+ T cell subpopulations showed that naive T cells and central memory T cells (TCM) decreased with declining CD4+ T cell counts in HIV-infected individuals, whereas the RA effector memory T cell subset (TEMRA) was expanded in HIV-infected individuals compared with controls. The TCM subset was found to have a significantly shorter lifespan in HIV-infected individuals with a high viral load than in uninfected individuals, and this population lost expression of IL-7Rα in more advanced disease. In contrast, the TEMRA population demonstrated a long half-life in HIV-infected individuals, accumulated as disease progressed, and lost expression of CD57. These data validate the use of the stable isotope/FACS/MS method to measure the kinetics of low-abundance human T cell populations in vivo. In addition, the data support the idea that TCM cells are the “true” memory CD8+ T cells important for protection against HIV progression and that the observed loss of these cells may be responsible for the loss of T cell memory activity in infected individuals.

The recent discovery of an important role for Th17 cells in the induction of experimental autoimmune encephalomyelitis (EAE) has led some to question the long-held belief that Th1 cells mediate this disease. Lees et al. (p. 8066 ) hypothesized that a small population of contaminating IL-17-producing cells in an adoptively transferred Th1 cell line would preferentially and quickly accumulate in the CNS following transfer. To test this hypothesis, the authors monitored the phenotype of T cells invading the CNS at different time points following adoptive transfer of a myelin oligodendrocyte glycoprotein (MOG)-specific, Th1-polarized cell line. Unexpectedly, IL-17-producing adoptively transferred cells were not found to preferentially accumulate in the CNS during the initiation, peak, or resolution phases of acute EAE. However, IL-17-producing host cells were recruited to the CNS early and were maintained throughout disease. These cells were heterogeneous, consisting of both γδ and αβ TCR+ T cells that were CD3+CD45+ and either CD4+CD8 or CD4+CD8+. Adoptive transfer of Th1 cells into IL-17−/− hosts resulted in amelioration of EAE, indicating that host production of IL-17 participates in disease pathogenesis. These data begin to clarify the relative roles of Th1 and Th17 cells in CNS autoimmunity.

Homeostatic proliferation of naive CD8+ T cells requires stimulation via IL-7 and the TCR, while that of memory CD8+ T cells requires IL-15. Recent data have indicated that stimulation with IL-21 in combination with either IL-7 or IL-15 can induce TCR-independent proliferation and effector functions in naive CD8+ T cells. Gagnon et al. (p. 7958 ) asked whether STAT3-utilizing cytokines other than IL-21 could affect CD8+ T cell activation and found that IL-6 could synergize with IL-7 or IL-15 to induce Ag-independent proliferation of naive or memory CD8+ T cells, respectively. Examination of the mechanism of cytokine synergy revealed that IL-6 or IL-21 induced STAT3 phosphorylation and augmented IL-7- or IL-15-mediated STAT5 phosphorylation and DNA binding. TCR-transgenic CD8+ T cells that were prestimulated with IL-7 or IL-15 together with IL-6 or IL-21 showed heightened sensitivity to Ag and augmented CTL activity upon subsequent TCR-mediated stimulation. CD8+ T cells stimulated with cytokines compared with those stimulated via the TCR were found to have distinct cell surface phenotypes that appeared to be independent of proliferative capacity. The authors propose that Ag-independent activation of CD8+ T cells by IL-6 and IL-7 or IL-15 may be involved in the transition from innate to adaptive immunity and may have implications for the development of autoimmunity.

The HIV-1 accessory protein Nef is important for viral pathogenesis and acts mainly via down-regulation of cell surface CD4, which leads to enhanced viral production and infectivity. The sorting signal responsible for Nef:CD4 internalization and subsequent intracellular trafficking has not been identified, so Jin et al. (p. 7878 ) assessed whether ubiquitination could play a role in Nef activity. Using an in vivo ubiquitination assay, the authors first determined that both HIV-1 Nef and SIV Nef could be ubiquitinated at multiple sites. The lysine residues of HIV-1 Nef were then substituted with arginine to determine which sites were ubiquitinated, and the main ubiquitin attachment sites were identified as K144, which was di-ubiquitinated, and K204, which was mono-ubiquitinated. A single point mutation at K144 completely prevented CD4 down-regulation, and exhaustive experiments confirmed that di-ubiquitination of this residue alone was critical for CD4 internalization. The identification of this mechanism for HIV-1-mediated CD4 down-regulation has implications for the understanding and future therapeutic modulation of HIV replication.

The CD2 family member 2B4, which is expressed on NK cells, γδ T cells, and memory CD8+ T cells, has been shown to both activate and inhibit NK cell functions in mice. The mechanism by which this single receptor mediates opposing functions is unclear, and it has not been established whether human 2B4 can also mediate inhibitory functions. Chlewicki et al. (p. 8159 ) therefore made a panel of 2B4-transfected cells to assess 2B4 activity in a controlled manner. Clones expressing human 2B4 or either of the two mouse 2B4 alleles could similarly mediate either activating or inhibitory functions. This activity generally correlated with 2B4 expression levels, such that lower surface expression favored activation and higher expression favored inhibition. The choice between activation and inhibition was also dependent upon the extent of 2B4 engagement; lower levels of cross-linking favored activation and higher levels favored inhibition. A third factor contributing to 2B4 function was expression of the adaptor molecule SLAM-associated protein (SAP); increasing levels of SAP favored activation over inhibition. In contrast, the adaptor molecules EAT-2A and EAT-2B did not appear to be required for inhibition. This clarification of 2B4 activity and its relation to three interrelated factors may have important implications for the understanding of NK cell activity and of multifunctional receptors in general.

Central memory CD4+ T cells (TCM) can protect against rechallenge with Leishmania major, and maintenance of immunity to this parasite requires the presence of IL-12. Pakpour et al. (p. 8299 ) analyzed the plasticity of L. major-specific CD4+ TCM to determine whether these TCM are predetermined to become Th1 effector cells. First, the authors determined that in vivo-generated Th1 cells and effector memory CD4+ T cells did not require IL-12 to produce IFN-γ. However, although either in vitro- or in vivo-generated CD63Lhigh TCM proliferated in the absence of IL-12, these cells could only become IFN-γ producers in the presence of IL-12. This suggested that these L. major-specific TCM were not committed to the Th1 lineage, an idea that was supported by the observation that very few of these cells expressed T-bet. Interestingly, TCM cells adoptively transferred into IL-12-deficient mice could become IL-4 producers, indicating that these cells had the potential to differentiate into either Th1 or Th2 effectors. These data suggest that L. major-specific TCM do not differentiate from effector Th1 cells and therefore require additional signals to fully differentiate into effector cells.

It has been shown that the CD8 coreceptor interacts with MHC to stabilize TCR:pMHC binding and to couple ligand binding to signaling initiation. To determine the structural basis for this interaction, Mallaun et al. (p. 8211 ) assessed the effect of deleting the TCR α-chain connecting peptide motif (α-CPM) on CD8 binding, T cell activation, and thymocyte differentiation. Transgenic mice bearing an α-CPM-deficient TCR showed a severe defect in thymic positive selection. This defect could be linked to a change in the CD8:MHC interaction that led to a reduced affinity of α-CPM mutant thymocytes for pMHC. The authors next analyzed the effect of α-CPM deficiency on TCR signaling and found that α-CPM mutant T cell hybridomas showed reduced TCR internalization compared with wild type and failed to produce IL-2 in response to either low- or high-affinity ligands. FRET and Western blot analysis then demonstrated that an intact α-CPM domain was required for CD3ζ:CD8β interaction and subsequent CD3ζ phosphorylation in response to low-affinity ligands, explaining the positive selection defect observed in mice lacking α-CPM. These data indicate the important role of α-CPM in TCR signaling, both in T cell activation and thymic selection.

Summaries written by Jennifer Hartt Meyers, Ph.D.