Recently developed major histocompatibility complex (MHC) multimer technologies allow visualization and isolation of antigen-specific T cells. However, functional analysis and in vivo transfer of MHC multimer-stained cells is hampered by the persistence of T-cell receptor (TCR) MHC interactions and subsequently induced signaling events. As MHC monomers do not stably bind to TCRs, we postulated that targeted disassembly of multimers into MHC monomers would result in dissociation of surface-bound TCR ligands. We generated a new type of MHC multimers, which can be monomerized in the presence of a competitor, resulting in rapid loss of the staining reagent. Following dissociation, the T cells are phenotypically and functionally indistinguishable from untreated cells. This 'reversible' T-cell staining procedure, which maintains the specificity and sensitivity of MHC multimer staining while preserving the functional status of T lymphocytes, may be of broad benefit for ex vivo investigation of T-cell functions and clinical applications.

Adoptive immunotherapy is a promising therapeutic approach for the treatment of chronic infections and cancer. Thereby, T cells within a certain range of high avidity for their cognate ligand are believed to be most effective. T cell receptor (TCR) transfer experiments indicate that a major part of avidity is hard-wired within the structure of the TCR. Unfortunately, rapid measurement of structural avidity of TCRs is difficult on living T cells. We developed a technology, where dissociation (koff-rate) of truly monomeric peptide major histocompatibility complex (pMHC) molecules bound to surface expressed TCRs can be monitored by real-time microscopy in a highly reliable manner. A first evaluation of this method on distinct human Cytomegalovirus (CMV) -specific T cell populations revealed unexpected differences in the koff-rates. CMV-specific T cells are currently being evaluated in clinical trials for efficacy in adoptive immunotherapy; therefore, determination of koff-rates could guide selection of the most effective donor cells. Indeed, in two different murine infection models, we demonstrate that T cell populations with lower koff-rates confer significantly better protection than populations with fast koff-rates. These data indicate that koff-rate measurements can improve the predictability of adoptive immunotherapy and provide diagnostic information on the in vivo quality of T cells.

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MRL/MpJ-Faslpr /J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8 ؉ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/ macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.

It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection. The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver) and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated interleukin-10 (IL-10) levels in plasma. Experiments using Il10 knockout mice, for which no differences between the susceptibilities of males and females to Listeria infection could be detected, confirmed the important role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences with regard to infection with Listeria monocytogenes and other intracellular pathogens have been reported for humans.Infectious diseases are a major cause of morbidity and mortality worldwide (52). It is well established that the sex of a host can significantly affect susceptibility to infection. A number of reports have shown that patients of one sex are more likely to get an infectious disease, and gender is often referred to as a risk factor for the severity and outcome of an illness; wellknown examples from human pathology include tuberculosis (13), sepsis (7), invasive amebiasis (1), toxoplasmosis-related entities (8, 42), and listeriosis (21). The underlying molecular mechanisms of this predisposition are largely unknown.Mice have been extensively used to study immune responses during infection, and it is not surprising that sex differences in susceptibility have also been observed in mouse models, further confirming clinical observations that female and male individuals handle infections differently. Most experimental settings examining a variety of different infectious agents have revealed a rather redundant susceptibility pattern, suggesting that female mice are in general more resistant to bacterial or viral diseases than males (4,17,27,30,35,36,53). In all these experimental models, increased resistance was associated with more vigorous and better-sustained immune responses in females (36,41,47,51), which have been attributed to hormoneregulated dissimilarities in immune cell function and cytokine production (5, 44). Nevertheless, the intricate mechanisms of sex differences in infection susceptibility have remained obscure.Listeria monocytogenes is an intracellular gram-positive bacterium that causes disease in immunocompromised individuals and pregnant women, often with deleterious consequences for the fetus (21). It is also one of the most widely used pathogens in exp...

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.The Dickkopf family of secreted proteins consists of four members, which share two conserved cysteine-rich domains (12,24). The hallmark of Dkk proteins is that they function as Wnt antagonists or agonists by binding to and inhibiting or activating the Wnt coreceptor LRP6 (1, 31, 45). They show regionalized expression during vertebrate embryogenesis (5,10,13,18,20,33,46). Dkk1 is the best-characterized member of the family. It acts as an embryonic head inducer, and when overexpressed it will induce extra heads in Xenopus and zebra fish (6,12,18,22,36,46). dkk1 mutant mice are embryonic lethal, and embryos lack anterior head structure and display fused digits (36). dkk2 mouse mutants are viable but show bone defects (28). Little is known about the biological role of dkk4.By a number of criteria, dkk3 appears as a divergent member of the dkk family. (i) By DNA sequence similarity, vertebrate dkk1, -2, and -4 are more related to each other than they are to dkk3 (12). (ii) Hydra has two dkk genes, one related to vertebrate dkk1, -2, and -4 (16) and one related to vertebrate dkk3 (9). This suggests an ancient phylogenetic separation between these family members, where dkk1, -2, and -4 but not dkk3 arose by gene duplication from an ancestral dkk (16). (iii) Soggy is a protein of unknown function with sequence similarity to dkk3 but not to other dkk genes (24). The similarity is most pronounced outside the two conserved Dkk cysteine-rich domains, raising the possibility that the gene arose from an ancestral dkk3 precursor. (iv) Unlike Dkk1, -2, and -4, Dkk3 does not act as a Wnt modulator (24,29,55). While all other tested Dkk proteins bind to and modulate the Wnt receptor LRP6, as well as the Dkk coreceptor Kremen, Dkk3 has no affinity to these transmembrane proteins (7,30,32,33), and no other proteins are known to interact with it.Like other dkk members, dkk3 is expressed during vertebrate development in suggestive patterns in many organs (7,33). Prominent expression of dkk3 is observed in the brain and in fibroblasts of adult rodents (17,24,34,37,56) and in the human adrenal cortex (50).

Many of the academic analyses calling Medellín’s development an “urban miracle” fall short with regard to discussion of the political economic implications of institutional shifts. An emerging transnational capitalist class promoted ‘good governance’ reforms and the embedding of neoliberalism in the urban context. Medellín’s neoliberal development agenda is not only a market-led strategy but also a particular form of hierarchic rule and distribution of power. Increased economic activities in the tertiary sector, the promotion of flexible labor markets, and the incorporation of the city into the global economy at the lower end of the value chain have not served as sustainable growth escalators for Medellín’s economy. The city continues to have high rates of un- and underemployment and is still the country’s most unequal city. These developments can by no means be described as miraculous. Muchos de los análisis académicos que describen el desarrollo de Medellín como un “milagro urbano” se quedan cortos en cuanto a la discusión de las implicaciones político-económicas de los cambios institucionales. Una nueva clase capitalista transnacional promovió las reformas de buena gobernanza y la incorporación del neoliberalismo en el contexto urbano. El plan de desarrollo neoliberal de Medellín no es sólo una estrategia orientada al mercado sino que también es una forma jerárquica de gobierno y distribución del poder. El aumento de las actividades económicas en el sector sectario, la promoción de un mercado laboral flexible y la incorporación de la ciudad en la economía global en el nivel más bajo de la cadena de valor no han servido como palancas para el crecimiento económico sustentable de Medellín. La ciudad sigue teniendo las tasas de desempleo y subempleo más altas de Colombia y todavía es la ciudad más desigual del país. Estos desarrollos no pueden ser descritos en modo alguno como milagrosos.

The activating receptor NKG2D recognizes a wide range of different ligands, some of which are primarily expressed in ''stressed'' tissues or on tumor cells. Until now, similar stimulatory effects on natural killer and CD8 ؉ T cells have been described for all NKG2D ligands, and the NKG2D receptor͞ligand system has therefore been interpreted as a sensor system involved in tumor immune surveillance and activation of immune responses. We show here that the NKG2D ligands H60 and MIC class 1 chain-related protein A (MICA) can also mediate strong suppressive effects on T cell proliferation. Responsiveness to H60-and MICA-mediated suppression requires IL-10 and involves a receptor other than NKG2D. These findings might provide explanations for the observation that strong in vivo NKG2D ligand expression, such as that on tumor cells, sometimes fails to support effective immune responses and links this observation to a distinct subgroup of NKG2D ligands.immunology ͉ NKG2D ligands