Cytokine expression by invariant natural killer T cells is tightly regulated throughout development and settings of type-2 inflammation.

Invariant natural killer T (iNKT) cells produce cytokines interleukin-4 (IL-4) and IL-13 during type-2 inflammatory responses. However, the nature in which iNKT cells acquire type-2 cytokine competency and the precise contribution of iNKT cell-derived IL-4 and IL-13 in vivo remains unclear. Using IL-13-reporter mice to fate-map cytokine-expressing cells in vivo, this study reveals that thymic iNKT cells express IL-13 early during development, and this IL-13-expressing intermediate gives rise to mature iNKT1, iNKT2, and iNKT17 subsets. IL-4 and IL-13 reporter mice also reveal that effector iNKT2 cells produce IL-4 but little IL-13 in settings of type-2 inflammation. The preferential production of IL-4 over IL-13 in iNKT2 cells results in part from their reduced GATA-3 expression. In summary, this work helps integrate current models of iNKT cell development, and further establishes non-coordinate production of IL-4 and IL-13 as the predominant pattern of type-2 cytokine expression among innate cells in vivo.

Visualization of immunotoxin-mediated tumor cell death in vivo.

We present a novel methodology to visualize tumor cells directly in a whole mouse. This technique combines immunohistochemistry with whole mouse sectioning. It lets one see the exact distribution of tumor cells throughout an animal and how effectively these cells are eliminated by cancer therapeutics. We used this technique to assess the efficacy of a T cell-specific immunotoxin in a severe combined immunodeficient mouse model of human T-cell leukemia. Severe combined immunodeficient mice were injected with one of two human T-cell acute lymphoblastic leukemia cell lines (Molt 3 and Molt 13) and were either left untreated or were treated with DA7, an immunotoxin specific for the T cell-associated antigen CD7. Mice were sacrificed after tumor cell injection and immunotoxin therapy, whole mouse cross-sections were prepared, and tumor cells in the sections were visualized by immunohistochemistry. No tumor cells were detected in DA7-treated mice injected with Molt 3, consistent with the long-term survival of this group and the sensitivity of Molt 3 to DA7 in vitro. In contrast, DA7 treatment did not visibly eliminate tumor cells in mice challenged with Molt 13, nor did it result in their long-term survival. Furthermore, tumor cells were detected in areas that may have otherwise been overlooked, and their distribution differed from that of mice injected with Molt 13 alone. These analyses indicate that whole mouse sectioning will be a valuable tool for assessing residual disease in the preclinical evaluation of cancer therapeutics.

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells.

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.

Visualizing the generation of memory CD4 T cells in the whole body.

It is thought that immunity depends on naive CD4 T cells that proliferate in response to microbial antigens, differentiate into memory cells that produce anti-microbial lymphokines, and migrate to sites of infection. Here we use immunohistology to enumerate individual naive CD4 T cells, specific for a model antigen, in the whole bodies of adult mice. The cells resided exclusively in secondary lymphoid tissues, such as the spleen and lymph nodes, in mice that were not exposed to antigen. After injection of antigen alone into the blood, the T cells proliferated, migrated to the lungs, liver, gut and salivary glands, and then disappeared from these organs. If antigen was injected with the microbial product lipopolysaccharide, proliferation and migration were enhanced, and two populations of memory cells survived for months: one in the lymph nodes that produced the growth factor interleukin-2, and a larger one in the non-lymphoid tissues that produced the anti-microbial lymphokine interferon-gamma. These results show that antigen recognition in the context of infection generates memory cells that are specialized to proliferate in the secondary lymphoid tissues or to fight infection at the site of microbial entry.

In vivo activation of antigen-specific CD4 T cells.

Physical detection of antigen-specific CD4 T cells has revealed features of the in vivo immune response that were not appreciated from in vitro studies. In vivo, antigen is initially presented to naïve CD4 T cells exclusively by dendritic cells within the T cell areas of secondary lymphoid tissues. Anatomic constraints make it likely that these dendritic cells acquire the antigen at the site where it enters the body. Inflammation enhances in vivo T cell activation by stimulating dendritic cells to migrate to the T cell areas and display stable peptide-MHC complexes and costimulatory ligands. Once stimulated by a dendritic cell, antigen-specific CD4 T cells produce IL-2 but proliferate in an IL-2--independent fashion. Inflammatory signals induce chemokine receptors on activated T cells that direct their migration into the B cell areas to interact with antigen-specific B cells. Most of the activated T cells then die within the lymphoid tissues. However, in the presence of inflammation, a population of memory T cells survives. This population is composed of two functional classes. One recirculates through nonlymphoid tissues and is capable of immediate effector lymphokine production. The other recirculates through lymph nodes and quickly acquires the capacity to produce effector lymphokines if stimulated. Therefore, antigenic stimulation in the presence of inflammation produces an increased number of specific T cells capable of producing effector lymphokines throughout the body.

Antigen-experienced CD4 T cells display a reduced capacity for clonal expansion in vivo that is imposed by factors present in the immune host.

It is thought that protective immunity is mediated in part by Ag-experienced T cells that respond more quickly and vigorously than naive T cells. Using adoptive transfer of OVA-specific CD4 T cells from TCR transgenic mice as a model system, we show that Ag-experienced CD4 T cells accumulate in lymph nodes more rapidly than naive T cells after in vivo challenge with Ag. However, the magnitude of clonal expansion by Ag-experienced T cells was much less than that of naive T cells, particularly at early times after primary immunization. Ag-experienced CD4 T cells quickly reverted to the slower but more robust clonal expansion behavior of naive T cells after transfer into a naive environment. Conversely, the capacity for rapid clonal expansion was acquired by naive CD4 T cells after transfer into passively immunized recipients. These results indicate that rapid in vivo response by Ag-experienced T cells is facilitated by Ag-specific Abs, whereas the limited capacity for clonal expansion is imposed by some other factor in the immune environment, perhaps residual Ag.

The effects of ultraviolet radiation on the moderate halophile Halomonas elongata and the extreme halophile Halobacterium salinarum.

Both the moderately halophilic bacterium, Halomonas elongata, and the extremely halophilic archaea, Halobacterium salinarum, can be found in hypersaline environments (e.g., salterns). On complex media, H. elongata grows over a salt range of 0.05-5.2 M, whereas, H. salinarum multiplies over a salt range of 2.5-5.2 M. The purpose of this study was to illustrate the effect that solar (UV-A and UV-B) and germicidal radiation (UV-C) had on the growth patterns of these bacteria at varied salt concentrations. Halomonas elongata grown on a complex medium at 0.05, 1.37, and 4.3 M NaCl was found to be more sensitive to UV-A and UV-B radiation, as the salt concentration of the medium increased. Halobacterium salinarum grown on a complex medium at 3.0 and 4.3 M NaCl did not show a significant drop in viability after 39.3 kJ.m-2 of UV-A and UV-B exposure. When exposed to UV-C, H. elongata exhibited substantially more sensitivity than H. salinarum. In H. elongata, differential sensitivity to UV-C was observed. At 0.05 M NaCl, H. elongata was less sensitive to UV-C than at 1.37 and 4.3 M NaCl. Both bacteria showed some photoreactivation when incubated under visible light following both UV-A, UV-B, and UV-C exposure. Mutagenesis following UV-C exposure was demonstrated by both organisms.