Population structure of Fusarium fujikuroi from California rice and water grass.

The recent observance of Fusarium fujikuroi, the causal agent of Bakanae disease of rice, in California provides a unique opportunity to assess the population diversity of an introduced pathogen in a new environment. We collected 172 isolates of this pathogen between 2000 and 2003 from California rice and two from water grass (Echinochloa spp.). Pathogenicity of F. fujikuroi was demonstrated on early water grass (E. oryzoides) and barnyard grass (E. crus-galli) indicating that weed control should be part of Bakanae management programs. Both mating types and six unique amplified fragment length polymorphism haplotypes corresponding to six identified vegetative compatibility groups were detected. The two most frequently isolated haplotypes encompassed 94% of the collected isolates, suggesting that clonal reproduction dominates. Coefficients of similarity between the unique haplotypes ranged from 0.94 to 0.98, and indicate that there is very little genotypic variation in the F. fujikuroi population in California. The near fixation of the MAT-1 idiomorph (observed ratio 170 MAT-1:4 MAT-2), is consistent with a hypothesis of predominant or exclusive asexual reproduction. The low level of introduced genotypic diversity, in conjunction with the asexual reproductive strategy of this population will slow evolutionary processes, including adaptation to the California environment.

Efficacy of an amitraz-impregnated collar in preventing transmission of Borrelia burgdorferi by adult Ixodes scapularis to dogs.

OBJECTIVE: To determine whether an amitraz-impregnated collar could prevent transmission of Borrelia burgdorferi by Ixodes scapularis to dogs. DESIGN: Laboratory trial. ANIMALS: 8 specific-pathogen-free Beagles. PROCEDURE: On days -15 and -1, all dogs had negative ELISA results for serum antibodies against B. burgdorferi. On day 0, 4 dogs were each fitted with an amitraz-impregnated (9%) collar, and 4 dogs served as untreated controls. On day 7, all dogs were infested with 100/scapularis (approx 50 females and 50 males) with a known B. burgdorferi infectivity rate of 39.4%. On days 21, 28, 35, 42, 56, 70, and 84, each dog was tested for serum antibodies against B. burgdorferi via ELISA and a western blot technique. Additional ELISA were also performed for serum antibodies against antigenically similar organisms. RESULTS: By day 70, all control dogs had developed serum ELISA responses ranging from 328 to 510 kinetics-ELISA units (equivalent to end-point titers of approx 43,500 to 60,000), whereas treated dogs remained seronegative throughout the study. Western blot assays performed on all serum samples confirmed that antibodies detected in control dogs reflected responses to specific antigens of B. burgdorferi, whereas treated dogs had no such antibodies. Additional serologic analyses confirmed that antibody responses observed in control dogs were not attributable to antigenically similar organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Amitraz-impregnated collars prevented transmission of B. burgdorferi in 4 of 4 treated dogs and may be a useful management tool for prevention of borreliosis in dogs.

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.

Lineage-specific requirement for signal transducer and activator of transcription (Stat)4 in interferon gamma production from CD4(+) versus CD8(+) T cells.

CD4(+) and CD8(+) T cells exhibit important differences in their major effector functions. CD8(+) T cells provide protection against pathogens through cytolytic activity, whereas CD4(+) T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-gamma, which can contribute to protective immunity. Here we show that CD4(+) and CD8(+) T cells differ in their regulation of IFN-gamma production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-gamma induced by interleukin (IL)-12/IL-18 signaling, but only CD4(+) T cells require Stat4 for IFN-gamma induction via the TCR pathway. In response to antigen, CD8(+) T cells can produce IFN-gamma independently of IL-12, whereas CD4(+) T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-gamma production based on differences in TCR signaling between CD4(+) and CD8(+) T cells.

From naive to memory. Development and regulation of CD4+ T cell responses.

Specific immune responses proceed through and are regulated at several stages: activation of naive cells and their differentiation into effector cells, completion of effector functions, development of memory cells, and subsequent reactivation of memory cells. To understand the development and regulation of CD4+ T cells in immune responses, naive CD4+ T cells were enriched from T cell receptor (TCR) transgenic mice, and used to generate effector and memory populations in vivo and in vitro. The expression of a common TCR on all of these developmental subsets has allowed us to compare directly their phenotype, cytokine profiles, activation requirements, and susceptibility to apoptosis. Our experiments have revealed interesting distinctions among naive, effector, and memory subsets of CD4+ T cells and have important implications for our understanding of immune responses.

In vivo persistence of CD8 polarized T cell subsets producing type 1 or type 2 cytokines.

Naive CD8 T cells can be polarized into effectors producing the type 1 cytokines IFN-gamma and IL-2 or the type 2 cytokines IL-4, IL-5, and IL-10, respectively. To study whether the polarized cytokine phenotype of the effectors is stable, we generated highly cytotoxic hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T cells producing type 1 or type 2 cytokines) effectors from Clone-4 TCR-transgenic mice, which were adoptively transferred into syngeneic adult thymectomized irradiated and bone marrow-reconstituted recipients. The highly activated blast-size, CD25+ Tc1 and Tc2 effectors gave rise to homogeneous resting CD25- CD44(high) Ly6C(high) Ag-specific populations, which persisted for at least 13 wk after adoptive transfer. These memory CD8 T cells, recovered 13 wk after transfer of Tc1 or Tc2 effectors, still produced either the type 1 or type 2 cytokines, i.e., IFN-gamma, or IL-4 and IL-5, respectively, upon restimulation with APCs loaded with the HA peptide, but not in the absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1 and Tc2 memory populations were comparable to that in memory CD4 cells, and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation. Thus, cytokine-polarized CD8 memory T cells are a source of a variety of cytokines, which were classically considered helper cytokines, opening new perspectives on their function as regulatory cells in an immune response.

Regulation of T cell subsets from naive to memory.

To gain insights into the development and regulation of immune responses, we have studied the phenotype, cytokine profiles, activation requirements, and susceptibility to apoptosis of naive CD4, Th1, Th2 polarized effectors, resting memory, and memory effector cells. T cell receptor (TCR) transgenic mice were utilized as a source of enriched naive cells that could be used to generate effector and memory populations. The direct comparison of these populations, which all bear the same TCR, has revealed some interesting distinctions. When restimulated with antigen, effector populations secrete high titers of cytokines in polarized patterns. Retaining their polarized profile, memory cells secrete low levels and memory effector cells secrete very large levels of cytokine. Unlike naive CD4 T cells, effector cell proliferation is not dependent on classic co-stimulation but does require a threshold level of TCR signaling that can be enhanced by accessory interactions. Memory cells have intermediate requirements for co-stimulation/accessory interactions. However, different thresholds of activation are required for production of various cytokines, with requirements for production of interleukin (IL) 2 >> interferon-gamma > IL-4. CD4 subsets also differ dramatically in their susceptibility to apoptosis. Naive Th2 effectors and resting memory cells undergo activation-induced cell death (AICD) 4-7 days after antigen stimulation. In contrast, both primary and memory Th1 effectors undergo rapid AICD mediated by Fas/FasL within 0.5-2 days after stimulation. AICD is substantially blocked by IL-2 and transforming growth factor-beta1, resulting in impressive effector expansion. The process of memory development from effector populations remains mysterious, but these studies suggest roles for cytokines in promoting survival.

Single cell analyses of cytokine production.

Subsets of T lymphocytes have been defined by their secretion of polarized patterns of cytokines. Type 1 and Type 2 subsets have been characterized in a variety of model systems both in vivo and in vitro and are a useful framework for studying immune responses. Recent technical advances have made it possible to analyze cytokine production by these populations at the level of individual cells.

Type 1 and type 2: a fundamental dichotomy for all T-cell subsets.

Cytokine secretion is not confined to CD4+ T cells; rather, Type 1 and Type 2 populations of CD8+ and gamma delta T cells can also be generated in vitro and isolated from in vivo situations. These subsets and their physiological functions are significant.

Relative perforin- and Fas-mediated lysis in T1 and T2 CD8 effector populations.

IFN-gamma-secreting T1 and IL-5-secreting T2 subsets of CD8 effectors were generated in vitro using freshly isolated cells from wild-type and perforin knockout mice stimulated with allogeneic Ag-presenting cells. Both T1 and T2 effectors from wild-type mice exhibited perforin-mediated cytolysis. T1, but not T2, populations from perforin knockout mice displayed significant lysis by the Fas-mediated pathway. Th1 cells have recently been shown to be regulated by Fas and we speculate that Fas-mediated mechanisms are involved in the regulation of both Th1 and T1 populations of T cells.

Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors.

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.

ATP is required for receptor-mediated endocytosis in intact cells.

We have demonstrated a requirement for cellular ATP in the receptor-mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric-transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I-BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.