The aged lymphoid tissue environment fails to support naïve T cell homeostasis.

Aging is associated with a gradual loss of naïve T cells and a reciprocal increase in the proportion of memory T cells. While reduced thymic output is important, age-dependent changes in factors supporting naïve T cells homeostasis may also be involved. Indeed, we noted a dramatic decrease in the ability of aged mice to support survival and homeostatic proliferation of naïve T cells. The defect was not due to a reduction in IL-7 expression, but from a combination of changes in the secondary lymphoid environment that impaired naïve T cell entry and access to key survival factors. We observed an age-related shift in the expression of homing chemokines and structural deterioration of the stromal network in T cell zones. Treatment with IL-7/mAb complexes can restore naïve T cell homeostatic proliferation in aged mice. Our data suggests that homeostatic mechanisms that support the naïve T cell pool deteriorate with age.

IL-7/anti-IL-7 mAb complexes restore T cell development and induce homeostatic T Cell expansion without lymphopenia.

IL-7, a member of the common gamma-chain family of cytokines, is essential for B and T lymphocyte development and homeostasis of mature T cell subsets. Thus, naive and memory T cells are both dependent on IL-7 for survival and homeostatic proliferation under lymphopenic conditions. In line with prior findings with IL-2, we show in this study that the biological activity of IL-7 in vivo is greatly increased by association with anti-IL-7 mAb. Under in vivo conditions, IL-7/mAb complexes displayed 50- to 100-fold higher activity than free IL-7 and induced massive expansion of pre-B cells. IL-7/mAb complexes also increased thymopoiesis in normal mice and restored thymopoeisis in IL-7-deficient mice. For mature T cells, IL-7/mAb complexes induced marked homeostatic proliferation of both naive and memory CD4(+) and CD8(+) cell subsets even under normal T cell-replete conditions. Finally, IL-7/mAb complexes were able to enhance the magnitude of the primary response of Ag-specific naive CD8(+) cells. The strong stimulatory activity of IL-7/mAb complexes could be useful for treatment of immunodeficiency and cancer.

The lymphopenic environment of CD132 (common gamma-chain)-deficient hosts elicits rapid homeostatic proliferation of naive T cells via IL-15.

Homeostatic proliferation for naive T cells is observed readily only under lymphopenic conditions in response to elevated levels of IL-7 and contact with self-MHC/peptide ligands. Homeostatic proliferation occurs at a slow pace and gradually induces the dividing cells to acquire characteristics of memory cells. We describe a novel type of homeostatic proliferation whereby naive T cells proliferate at a significantly faster rate, resembling the proliferation speed induced by foreign Ags, and the expanding cells rapidly differentiate into central memory cells. Remarkably, such rapid homeostatic proliferation is driven by a combination of IL-2 and IL-15, with IL-15 playing a bigger role, and applies for a wide repertoire of CD8(+) naive T cells, including many TCR-transgenic lines, even those that fail to undergo IL-7-driven homeostatic proliferation. Thus, naive T cells can be induced to undergo homeostatic proliferation of variable speed with a few members of the common gamma-chain (CD132) family of cytokines, the speed of proliferation depending on the levels of the particular cytokine involved.

Disorder and intermolecular interactions in a family of tetranuclear Ni(II) complexes probed by high-frequency electron paramagnetic resonance.

High-frequency electron paramagnetic resonance (HFEPR) data are presented for four closely related tetranuclear Ni(II) complexes, [Ni(hmp)(MeOH)Cl]4.H2O (1a), [Ni(hmp)(MeOH)Br]4.H2O (1b), [Ni(hmp)(EtOH)Cl]4.H2O (2), and [Ni(hmp)(dmb)Cl]4 (3) (where hmp(-) is the anion of 2-hydroxymethylpyridine and dmb is 3,3'-dimethyl-1-butanol), which exhibit magnetic bistability (hysteresis) and fast magnetization tunneling at low temperatures, properties which suggest they are single-molecule magnets (SMMs). The HFEPR spectra confirm spin S = 4 ground states and dominant uniaxial anisotropy (DSz(2), D < 0) for all four complexes, which are the essential ingredients for a SMM. The individual fine structure peaks (due to zero-field splitting) for complexes 1a, 1b, and 2 are rather broad. They also exhibit further (significant) splitting, which can be explained by the fact that there exists two crystallographically distinct Ni 4 sites in the lattices for these complexes, with associated differences in metal-ligand bond lengths and different zero-field splitting (ZFS) parameters. The broad EPR lines, meanwhile, may be attributed to ligand and solvent disorder, which results in additional distributions of microenvironments. In the case of complex 3, there are no solvate molecules in the structure, and only one distinct Ni 4 molecule in the lattice. Consequently, the HFEPR data for complex 3 are extremely sharp. As the temperature of a crystal of complex 3 is decreased, the HFEPR spectrum splits abruptly at approximately 46 K into two patterns with very slightly different ZFS parameters. Heat capacity data suggest that this is caused by a structural transition at 46.6 K. A single-crystal X-ray structure at 12(2) K indicates large thermal parameters on the terminal methyl groups of the dmb (3,3-dimethyl-1-butanol) ligand. Most likely there exists dynamic disorder of parts of the dmb ligand above 46.6 K; an order-disorder structural phase transition at 46.6 K then removes some of the motion. A further decrease in temperature (<6 K) leads to further fine structure splittings for complex 3. This behavior is thought to be due to the onset of short-range magnetic correlations/coherences between molecules caused by weak intermolecular magnetic exchange interactions.

An intense form of homeostatic proliferation of naive CD8+ cells driven by IL-2.

In conditions of T lymphopenia, interleukin (IL) 7 levels rise and, via T cell receptor for antigen-self-major histocompatibility complex (MHC) interaction, induce residual naive T cells to proliferate. This pattern of lymphopenia-induced "homeostatic" proliferation is typically quite slow and causes a gradual increase in total T cell numbers and differentiation into cells with features of memory cells. In contrast, we describe a novel form of homeostatic proliferation that occurs when naive T cells encounter raised levels of IL-2 and IL-15 in vivo. In this situation, CD8(+) T cells undergo massive expansion and rapid differentiation into effector cells, thus closely resembling the T cell response to foreign antigens. However, the responses induced by IL-2/IL-15 are not seen in MHC-deficient hosts, implying that the responses are driven by self-ligands. Hence, homeostatic proliferation of naive T cells can be either slow or fast, with the quality of the response to self being dictated by the particular cytokine (IL-7 vs. IL-2/IL-15) concerned. The relevance of the data to the gradual transition of naive T cells into memory-phenotype (MP) cells with age is discussed.

Better understanding of the species with the shortest Re2(6+) bonds and related Re2)(7+) species with tetraguanidinate paddlewheel structures.

A series of compounds has been made containing quadruply bonded Re2(hpp)4X2 species (hpp = the anion of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2a]pyrimidine), where X is CF3SO3 (1), CF3CO2 (2), and F (3). The distances of 2.1562(7), 2.1711(5), and 2.1959(4) A for 1-3 show significant effects of the sigma and pi electron donating ability of the axial ligands on the metal-metal distance. With the weakly coordinating triflate ligand the Re-Re distance is the shortest for any quadruple bonded species known. In addition to examining the effects of axial ligands on the Re2(hpp)42+ core, our study of the Re2(hpp)43+ core is being extended beyond the preliminary results previously reported in only one compound [Re2(hpp)4Cl2]PF6 (Dalton Trans. 2003, 1218). We now report the structural characterization by both X-ray and neutron diffraction of the compound [Re2(hpp)4F](TFPB)2, 4 (TFPB = the anion tetrakis[3,5-bis(trifluoromethyl)phenyl]borate), and a detailed study by EPR spectroscopy of [Re2(hpp)4Cl2]PF6 at 9.5, 34.5, and 95 GHz frequencies, using dilute fluid solutions, frozen glass, and neat powder, show that the unpaired electron in the [Re2(hpp)4Cl2]+ ion is in an MO of predominant metal character with little mixing from the guanidinate ligands.

Aire-deficient mice develop hematopoetic irregularities and marginal zone B-cell lymphoma.

Autoimmune polyendocrine syndrome type I (APS I) is an inherited recessive disorder with a progressive immunological destruction of many tissues including the adrenal cortex, the parathyroid glands, and the gonads. APS I is caused by mutations in the AIRE gene (autoimmune regulator), expressed in cells of the thymus and spleen, suggesting a role in central and peripheral tolerance. Aire(-/-) mice replicate the autoimmune features of APS I patients with the presence of multiple autoantibodies and lymphocytic infiltrates in various tissues, but young mice appear clinically healthy. We here report the investigation of 15- to 24-month-old Aire(-/-) mice. We did not observe any endocrinological abnormalities, nor did sera from these mice recognize known APS I autoantigens. Interestingly, however, there was a high frequency of marginal zone B-cell lymphoma in Aire(-/-) mice and liver infiltrates of B cells, suggesting chronic antigen exposure and exaggerated activation. Furthermore, increased numbers of monocytes in blood were identified as well as augmented numbers of metallophilic macrophages in the spleen. We propose that Aire, in addition to its function in the thymus, also has a peripheral regulatory role by controlling the development of antigen-presenting cells (APCs) and marginal zone B-cell activation.

Observation of a half step magnetization in the {Cu3}-type triangular spin ring.

We report pulsed field magnetization and ESR experiments on a {Cu3} nanomagnet, where antiferromagnetically coupled Cu(2+) (S = 1/2) ions form a slightly distorted triangle. The remarkable feature is the observation of a half step magnetization, hysteresis loops, and an asymmetric magnetization between a positive and a negative field in a fast sweeping external field. This is attributed to an adiabatic change of magnetization. The energy levels determined by ESR unveil that the different mixing nature of a spin chirality of a total S = 1/2 Kramers doublet by virtue of Dzyaloshinskii-Moriya interactions is decisive for inducing half step magnetization.

Increased antigen presenting cell-mediated T cell activation in mice and patients without the autoimmune regulator.

Patients with autoimmune polyendocrine syndrome type I (APS I)suffer from endocrine and non-endocrine disorders due to mutations in the autoimmune regulator gene (AIRE). Mouse Aire is expressed both in thymic medullary epithelial cells and in peripheral antigen-presenting cells, suggesting a role in both central and peripheral tolerance. We here report that Aire(-/-) dendritic cells (DC) activate naive T cells more efficiently than do Aire(+/+) DC. Expression array analyses of Aire(-/-) DC revealed differential regulation of 68 transcripts, among which, the vascular cell adhesion molecule-1 (VCAM-1) transcript was up-regulated in Aire(-/-) DC. Concurrently, the expression of the VCAM-1 protein was up-regulated on both Aire(-/-) DC and monocytes from APS I patients. Blocking the interaction of VCAM-1 prevented enhanced Aire(-/-) DC stimulation of T cell hybridomas. We determined an increased number of DC in spleen and lymph nodes and of monocytes in the blood from Aire(-/-) mice, and an increased number of blood monocytes in APS I patients. Our findings imply a role for Aire in peripheral DC regulation of T cell activation, and suggest that Aire participates in peripheral tolerance.

Proof of large positive zero-field splitting in a Ru2 5+ paddlewheel.

We present the synthesis, as well as the structural and magnetic characterization, of [Ru2(D(3,5-Cl2Ph)F)4Cl(0.5H2O)].C6H14 (D(3,5-Cl2Ph)F = N,N'-di(3,5-dichlorophenyl)formamidinate), a Ru2(5+) compound having a 4B(2u) ground state derived from a sigma2pi4delta2pi2delta electron configuration. The persistence of this configuration from 27 to 300 K is shown by the invariance of the Ru-Ru distance. Orientation-dependent magnetic susceptibility (chiT) and magnetization (M(H)) data are in accord with a spin quartet ground state with large magnetocrystalline anisotropy associated with a large axial zero-field splitting (D) parameter. Theoretical fits to chiT and M(H) plots yielded D/kB = +114 K, implying an S = +/-1/2 Kramers doublet ground state at low temperature. Single-crystal and powder EPR data are consistent with this result, as the only observed transition is between the M(s) = +/1/2 Zeeman levels. The g values are g(perpendicular) = 2.182, g(parallel) = 1.970, and D = 79.8 cm(-1). The totality of the results demands D >> 0.

Recent immune status determines the source of antigens that drive homeostatic T cell expansion.

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.

Crystalline and water soluble Cr(4+) and Cr(5+) model compounds for chromium toxicity studies.

We discuss two complexes of Cr(4+) for their possible utility as models for Cr toxicity studies. They are Cr(dien)(O2)2(.)H2O (dien = diethylenetriamine) and Cr(NH3)3(O2)2, which have been recently characterized by x-ray diffraction and magnetic techniques. We present their optical and infrared absorption spectra as quick aids in their identification procedure. We also summarize the general properties of some well-characterized Cr(5+) compounds. All of these compounds are water soluble with the Cr(5+) samples being fairly stable in basic solutions. They can all be prepared as pure crystals with a shelf life of over 2 years when stored in a refrigerator.

Observation of symmetry lowering and electron localization in the doublet-states of a spin-frustrated equilateral triangular lattice: Cu3(O2C16H23) x 1.2C6H12.

Cu3(O2C16H23)6.1.2C6H12, containing a Cu36+ core in an equilateral triangle geometry, has been found to be a versatile model system for investigating the spin-frustration phenomenon in a triangular lattice. It affords well-resolved EPR spectra from both of the two possible (Stotal = 1/2 and 3/2) spin states of the Cu36+ core. From 295 to 100 K, the spectra consist of a triplet, but with the central line overlapped by an additional, sharp peak, which replaces the triplet at 30 K and below. The triplet was thus assigned to the excited state with Stotal = 3/2, located at 324 +/- 5 K ( approximately 225 cm-1), with the zero-field parameters D = -535 G, E = 0, g parallel = 2.209 and g perpendicular = 2.057. The singlet was attributed to the Stotal = 1/2 state, with gxx = 2.005, gyy = 2.050, gzz = 2.282, and, surprisingly, a hyperfine splitting arising from a single Cu2+ nucleus, with Azz = 157 G. The detailed magnetic measurements on a three-electron, equilateral triangular system, and the observation of symmetry lowering in the doublet ground state, should be of broad theoretical and experimental interest in molecular magnetism.

Applications of high-field (W-band) EPR to M-M bonded units (M = Cr, Mo): the first confirmed oxidation of a Cr2 4+ paddlewheel complex to a stable isostructural Cr2 5+ product.

The EPR spectra of [Cr(2)[(PhN)(2)CN(CH(2))(4)](4)]PF(6) and [Mo(2)(TiPB)(4)]PF(6) (TiPB = anion of 2,4,6-triisopropylbenzoic acid) at W-band are shown to have g values significantly lower than 2.00 and exhibit parallel and perpendicular components (not resolved at X-band). Therefore the unpaired electrons of the M(2)(5+) units must reside on metal-based (not ligand-based) orbitals. Thus, the chromium compound must be considered as the first confirmed oxidation product of a Cr(2)(4+) paddlewheel complex comparable to the Mo(2)(5+) compounds.

Systematic mutagenesis of the functional domains of AIRE reveals their role in intracellular targeting.

Mutations in the human autoimmune regulator (AIRE ) gene cause a multi-systemic autoimmune syndrome that is known as autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). To date more than 39 different disease mutations have been identified. They span the entire region of the AIRE gene that encodes a polypeptide with multiple functional domains: an N-terminal homogeneously staining region (HSR), a bipartied nuclear localization signal (NLS), a SAND domain, two PHD fingers and four nuclear receptor targeting motifs. The APECED mutations include insertions, deletions, substitutions and introduction of premature termination codons, while most mutations disrupt one of the functional domains. We have constructed a series of deletion mutants systematically removing one or more functional domain(s) and investigated the stability and sub-cellular compartmentalization of the corresponding polypeptides. Here we show that the first 188 amino acids, containing the HSR domain and the NLS proved necessary for both cytoplasmic filament formation and nuclear targeting. Deletion of the SAND domain and even point mutations in the SAND domain, resulted in the aggregation of the polypeptides in the cytoplasm and interfered with the proper nuclear targeting. The PHD fingers seemed to be necessary for the formation of characteristic dot-like complexes in the nucleus, but their deletion did not interfere with nuclear entry.

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

Aire deficient mice develop multiple features of APECED phenotype and show altered immune response.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal recessive disease caused by mutations in the AIRE gene. Here we have produced knock-out mice for the Aire gene. The Aire-/- mice develop normally; however, autoimmune features of APECED in Aire-/- mice are evident, including multiorgan lymphocytic infiltration, circulating autoantibodies and infertility. The distribution of B and T cells and thymic maturation as well as activation of T cells appear normal, while the TCR-Vbeta repertoire is altered in peripheral T cells of Aire-/- mice. When mice are challenged with immunization, the peripheral T cells of Aire-/- mice have a 3-5-fold increased proliferation. These findings suggest that the Aire gene is not necessary for normal T cell education and development, while a defect in immune response detected in challenged Aire-/- mice underlines the crucial role of AIRE/Aire in maintaining homeostatic regulation in the immune system.