Spontaneous mutation in the Cd79b gene leads to a block in B-lymphocyte development at the C' (early pre-B) stage.

We discovered B-lymphocyte-deficient mice within a group of B10.A-CD45.1 mice, and established that this deficiency was a recessively inherited trait. Gene mapping and sequence analysis showed a mutation in the third exon of the Cd79b gene (c.224G>A) that leads to the generation of a stop codon (W75X) in the mutant mouse. Fluorescent-activated cell sorting analysis of bone marrow cells showed that the mutant mice did not express the CD79B antigen. To establish where the block in development happens, we analyzed CD43(pos)B220(pos) B-lymphocyte precursors present in the mutant mice and found that the fraction C' (corresponding to early pre-B lymphocytes) was absent in the mutant mouse, whereas fractions B and C showed a relative accumulation. As expected, we found no IgG or IgA in mutant mice. These results suggest that this CD79b-mutant strain may be a useful tool for immunological research in human immunodeficiencies.

T-cell control of IL-12p75 production.

It is currently thought that IL-12, produced by dendritic cells (DC) early after stimulation by bacterial pathogens or lipopolysaccharide (LPS), acts as a pro-inflammatory cytokine bridging the innate and adaptive immune responses. We found, however, that it is only the p40 subunit and not the IL-12p75 heterodimer that is secreted early in copious amounts in response to LPS. Neither naïve T cells, nor a variety of microbial products, were able to induce IL-12p75 production unless the DC were conditioned by the presence of interferon-gamma (IFN-gamma) or by encounter with previously activated T cells. The inability of naïve T cells or of bacterial products to induce IL-12 argues against its early role as the initiator of innate and adaptive immune responses.

The role of dendritic cells, B cells, and M cells in gut-oriented immune responses.

Although induction of T cell responses to fed Ag (oral tolerance) is thought to happen within the organized lymphoid tissue of the gut, we found that mice lacking Peyer's patches, B cells, and the specialized Ag-handling M cells had no defect in the induction of T cell responses to fed Ag, whether assayed in vitro by T cell proliferation or cytokine production, or in vivo by delayed-type hypersensitivity or bystander suppression against mycobacterial Ags in CFA. Feeding of Ag had a major influence on dendritic cells from fed wild-type or muMT mice, such that these APCs were able to elicit a different class of response from naive T cells in vitro. These results suggest that systemic immune responses to soluble oral Ags do not require an organized gut-associated lymphoid tissue but are most likely induced by gut-conditioned dendritic cells that function both to initiate the gut-oriented response and to impart the characteristic features that discriminate it from responses induced parenterally.

Testing time-, ignorance-, and danger-based models of tolerance.

In this study, we present data showing that tolerance to Ags in the periphery is not determined by the time at which the Ag appears, or by special properties of tissues in newborn mice or newly developing immune systems. We placed male grafts onto immunoincompetent female mice, allowed the grafts to heal for up to 5 mo, and then repopulated the recipients with fetal liver stem cells. We found that the newly arising T cells were neither tolerant nor ignorant of the grafts, but promptly rejected them, though they did not reject female grafts, nor show any signs of autoimmunity. We also found that the H-Y Ag was continuously cross-presented on host APCs, that this presentation was immunogenic, not tolerogenic, and that it depended on the continuous presence of the graft. In searching for the stimulus that might activate the host APCs, we analyzed mRNA expression with a highly sensitive real-time quantitative PCR assay. By using two different "housekeeping" molecules for comparison, we analyzed the message levels for several stress and/or inflammatory molecules in the healed grafts. We found that the long-healed grafts were not equivalent to "normal" skin because the healed grafts expressed lower levels of GAPDH. Altogether, these data suggest that acceptance vs rejection of peripheral tissues is not attributable to ignorance, timing-based tolerance, or special circulation properties of naive T cells in neonatal tissues. It is more likely attributable to an aspect of the context of Ag presentation that remains to be identified.

Immunity or tolerance: opposite outcomes of microchimerism from skin grafts.

Solid organ transplants contain small numbers of leukocytes that can migrate into the host and establish long-lasting microchimerism. Although such microchimerism is often associated with graft acceptance and tolerance, it has been difficult to demonstrate a true causal link. Using skin from mutant mice deficient for leukocyte subsets, we found that donor T-cell chimerism is a 'double-edged sword' that can result in very different outcomes depending on the host's immunological maturity and the antigenic disparities involved. In immunologically mature hosts, chimerism resulted in immunity and stronger graft rejection. In immature hosts, it resulted in tolerance to the chimeric T cells, but not to graft antigens not expressed by the chimeric cells. Clinical efforts aimed at augmenting chimerism to induce tolerance must take into account the maturation state of host T cells, the type of chimerism produced by each organ and the antigenic disparities involved, lest the result be increased rejection rather than tolerance.

Danger signals: SOS to the immune system.

The activation of dendritic cells, necessary for the initiation of primary and secondary immune responses, can be induced by endogenous danger signals - released by tissues undergoing stress, damage or abnormal death - and also by exogenous danger signals elaborated by pathogens. Some endogenous danger signals that recently have been discovered are heat-shock proteins, nucleotides, reactive oxygen intermediates, extracellular-matrix breakdown products, neuromediators and cytokines like the IFNs. We propose that allergy may be initiated by the direct damage of dendritic or other cells by toxic chemicals and allergenic proteases, and suggest that the triggering of danger signal receptors by exogenous pathogen-derived molecules may be more to the advantage of the pathogen than to the host.

Gamma chain required for naïve CD4+ T cell survival but not for antigen proliferation.

Lymphoid homeostasis is required to ensure immune responsiveness and to prevent immunodeficiency. As such, the immune system must maintain distinct populations of naïve T cells that are able to respond to new antigens as well as memory T cells specific to those antigens it has already encountered. Though both naïve and memory T cells reside in and traffic through secondary lymphoid organs, there is growing evidence that the two populations may be regulated differently. We show here that naïve T cell survival and memory T cell survival have different requirements for cytokines (including the interleukins IL-2, IL-4, IL-7, IL-9 and IL-15) that use the common cytokine receptor gamma chain (gamma c). Using monoclonal populations of antigen-specific CD4+ T cells, we found that naïve T cells cannot survive without gamma c, whereas memory T cells show no such requirement. In contrast, neither naïve nor gamma c-deficient memory T cells were impaired in their ability to proliferate and produce cytokines in response to in vivo antigenic stimulation. These data call into question the physiological role of gamma c-dependent cytokines as T cell growth factors and show that naïve and memory CD4+ T cell survival is maintained by distinct mechanisms.

Natural adjuvants: endogenous activators of dendritic cells.

Dendritic cells, the most potent antigen-presenting cells, need to be activated before they can function to initiate an immune response. We report here that, in the absence of any foreign substances, dendritic cells can be activated by endogenous signals received from cells that are stressed, virally infected or killed necrotically, but not by healthy cells or those dying apoptotically. Injected in vivo with an antigen, the endogenous activating substances can function as natural adjuvants to stimulate a primary immune response, and they may represent the natural initiators of transplant rejection, spontaneous tumor rejection, and some forms of autoimmunity.

On the lifespan of virgin T lymphocytes.

To study the lifespan of virgin T lymphocytes, we removed the thymus from adult female mice and then, at various times afterward, tested their ability to mount an immune response to a newly encountered Ag, the male Ag H-Y. We found that unprimed thymectomized mice were able to generate a primary response to H-Y for some time after thymectomy but lost this ability at approximately 6 mo. In contrast, mice that were primed to H-Y just after thymectomy continued to display immunological memory to H-Y for >1 year. These experiments show that primary immune responses disappear in the absence of a thymus.

Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion.

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

Use of transgenic cell lines in mechanistic studies of drug metabolism.

Heterologously expressed human drug-metabolizing enzyme systems in a variety of hosts (mammalian cell lines, baculovirus/insect cells, yeast and Escherichia coli) have their distinct advantages for particular studies of biotransformation or mechanistic processes. In contrast to classical in vitro systems such as tissue slices, hepatocytes or subcellular fractions, expressed enzymes allow the study of single enzyme reactions in isolation. Furthermore, metabolic reactions of enzymes expressed only in minor amounts in human tissue can be assessed. Here we present an overview of how recombinant enzymes are being used in biotransformation studies and we will present several examples of applications of recombinant cytochrome P450 preparations during drug development. It is beyond the scope of this overview to describe all experimental procedures in detail since they are based on published techniques unless otherwise indicated. Examples of applications of recombinant cytochrome P450 preparations include the involvement of human metabolizing enzymes in a metabolic pathway and their metabolic products, mechanistic studies to determine specific drug-drug interactions at the metabolic level and detection of mechanism-based inactivation of drug-metabolizing enzymes. Heterologous expression systems offer a constant and reproducible source of human drug-metabolizing enzymes that are easily available by standard laboratory techniques. Considering the constraints on the availability and use of human tissue it is likely that these systems will be widely used in the future. But, because of the unequal distribution of individual drug-metabolizing enzymes in man, extrapolation needs to be done carefully.