Jeannine M. Durdik

Inducible nitric oxide synthase in T cells regulates T cell death and immune memory.

The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS(+/+) APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS(-/-) T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.

Pentoxifylline Functions As an Adjuvant In Vivo to Enhance T Cell Immune Responses by Inhibiting Activation-Induced Death

Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants.

Comparison of microdialysis sampling perfusion fluid components on the foreign body reaction in rat subcutaneous tissue.

Microdialysis sampling is a commonly used technique for collecting solutes from the extracellular space of tissues in laboratory animals and humans. Large molecular weight solutes can be collected using high molecular weight cutoff (MWCO) membranes (100kDa or greater). High MWCO membranes require addition of high molecular weight dextrans or albumin to the perfusion fluid to prevent fluid loss via ultrafiltration. While these perfusion fluid additives are commonly used during microdialysis sampling, the tissue response to the loss of these compounds across the membrane is poorly understood. Tissue reactions to implanted microdialysis sampling probes containing different microdialysis perfusion fluids were compared over a 7-day time period in rats. The base perfusion fluid was Ringers solution supplemented with either bovine serum albumin (BSA), rat serum albumin (RSA), Dextran-70, or Dextran-500. A significant inflammatory response to Dextran-70 was observed. No differences in the tissue response between BSA and RSA were observed. Among these agents, the BSA, RSA, and Dextran-500 produced a significantly reduced inflammatory response compared to the Dextran-70. This work demonstrates that use of Dextran-70 in microdialysis sampling perfusion fluids should be eliminated and replaced with Dextran-500 or other alternatives.

Apoptosis-Inducing Factor Regulates Death in Peripheral T Cells

Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein with multiple roles in apoptosis as well as in cellular respiration and redox regulation. The harlequin (Hq) mouse strain carries an aif locus modification causing reduced Aif expression. We demonstrate that activated CD4+ and CD8+ peripheral T cells from Hq mice show resistance to neglect-induced death (NID) triggered by growth factor withdrawal, but not to death induced by multiple agents that trigger DNA damage. Aif translocates to the nucleus in cells undergoing NID, and, in Hq T cell blasts, resistance to NID is associated with reduced cytosolic release of mitochondrial cytochrome c, implicating Aif in this event. In contrast, Hq T cell blasts express higher levels of CD95L, demonstrating increased susceptibility to activation-induced cell death (AICD) and apoptosis triggered by hydrogen peroxide. Superoxide scavenging protects from AICD in wild-type, but not Hq, T cell blasts, suggesting that Aif plays a crucial superoxide-scavenging role to regulate T cell AICD. Finally, the altered pattern of death susceptibility is reproduced by siRNA-mediated reduction of Aif expression in normal T cells. Thus, Aif serves nonredundant roles, both proapoptotic and antiapoptotic, in activated peripheral T cells.

Localized delivery of dexamethasone-21-phosphate via microdialysis implants in rat induces M(GC) macrophage polarization and alters CCL2 concentrations

Microdialysis sampling probes were implanted into the subcutaneous space on the dorsal side of male Sprague Dawley rats to locally deliver dexamethasone-21-phosphate (Dex) with the aim of altering in vivo macrophage polarization. Macrophage polarization is of significant interest in the field of biomaterials since wound-healing macrophages are a possible means to extend implant life as well as improve tissue remodeling to an implant. Quantitative analysis of CCL2 in collected dialysates, gene expression and immunohistochemistry performed on the tissue surrounding the microdialysis implant were used to evaluate if Dex polarized macrophages. Dex infusion down-regulated IL-6 and CCL2 gene expression and decreased CCL2 concentrations in dialysates collected at the implant site. Dex appeared to have no significant effect on the gene regulation of CD163, a commonly used M2c macrophage surface marker; Arg2; and iNOS2. However, Dex infusion was effective at increasing the number of CD163(+) cells surrounding the implanted microdialysis probe. This work demonstrates the use of microdialysis sampling to deliver agents such as Dex to alter macrophage polarization in vivo while allowing the ability to collect cytokines in the surrounding microenvironment.

A role for apoptosis-inducing factor in T cell development

The mitochondrial flavoprotein Aif facilitates murine thymocyte development by reducing oxidative stress.

Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function

BackgroundAs individual naïve CD4 T lymphocytes circulate in the body after emerging from the thymus, they are likely to have individually varying microenvironmental interactions even in the absence of stimulation via specific target recognition. It is not clear if these interactions result in alterations in their activation, survival and effector programming. Naïve CD4 T cells show unimodal distribution for many phenotypic properties, suggesting that the variation is caused by intrinsic stochasticity, although underlying variation due to subsets created by different histories of microenvironmental interactions remains possible. To explore this possibility, we began examining the phenotype and functionality of naïve CD4 T cells differing in a basic unimodally distributed property, the CD4 levels, as well as the causal origin of these differences.ResultsWe examined separated CD4hi and CD4lo subsets of mouse naïve CD4 cells. CD4lo cells were smaller with higher CD5 levels and lower levels of the dual-specific phosphatase (DUSP)6-suppressing micro-RNA miR181a, and responded poorly with more Th2-skewed outcomes. Human naïve CD4lo and CD4hi cells showed similar differences. Naïve CD4lo and CD4hi subsets of thymic single-positive CD4 T cells did not show differences whereas peripheral naïve CD4lo and CD4hi subsets of T cell receptor (TCR)-transgenic T cells did. Adoptive transfer-mediated parking of naïve CD4 cells in vivo lowered CD4 levels, increased CD5 and reactive oxygen species (ROS) levels and induced hyporesponsiveness in them, dependent, at least in part, on availability of major histocompatibility complex class II (MHCII) molecules. ROS scavenging or DUSP inhibition ameliorated hyporesponsiveness. Naïve CD4 cells from aged mice showed lower CD4 levels and cell sizes, higher CD5 levels, and hyporesponsiveness and Th2-skewing reversed by DUSP inhibition.ConclusionsOur data show that, underlying a unimodally distributed property, the CD4 level, there are subsets of naïve CD4 cells that vary in the time spent in the periphery receiving MHCII-mediated signals and show resultant alteration of phenotype and functionality via ROS and DUSP activity. Our findings also suggest the feasibility of potential pharmacological interventions for improved CD4 T cell responses during vaccination of older people via either anti-oxidant or DUSP inhibitor small molecules.

Increase in double-stranded DNA break-related foci in early-stage thymocytes of aged mice

Cellular and molecular mechanisms involved in aging are notoriously complex. Aging-related immune decline of T lymphocyte function is partly caused by attrition of thymic T cell development, which involves programmed creation and repair of DNA breaks for generating T cell receptors. Aging also leads to significant alterations in the cellular DNA repair ability. We show that higher levels of gamma-phosphorylated H2AX (pH2AX), which marks DNA double-stranded breaks (DSBs), were detectable in early thymocyte subsets of aged as compared to young mice. Also, while only 1-2 foci of nuclear accumulation of pH2AX were detectable in these early thymocytes from young mice, cells from aged mice showed higher numbers of pH2AX foci. In CD4-CD8- double-negative (DN) thymocytes of aged mice, which showed the highest levels of DSBs, there was a modest increase in levels of the DNA repair protein MRE11, but not of either Ku70, another DNA repair protein, or the cell cycle checkpoint protein p53. Thus, immature thymocytes in aged mice show a marked increase in DNA DSBs with only a modest enhancement of repair processes, and the resultant cell cycle block could contribute to aging-related defects of T cell development.

Effects of delayed delivery of dexamethasone-21-phosphate via subcutaneous microdialysis implants on macrophage activation in rats.

Macrophage activation is of interest in the biomaterials field since macrophages with an M(Dex) characteristic phenotype, i.e., CD68(+)CD163(+), are believed to result in improved integration of the biomaterial as well as improved tissue remodeling and increased biomaterial longevity. To facilitate delivery of a macrophage modulator, dexamethasone-21-phosphate (Dex), microdialysis probes were subcutaneously implanted in male Sprague-Dawley rats. Dex localized delivery was delayed to the third day post implantation as a means to alter macrophage activation state at an implant site. To better elucidate the molecular mechanisms associated with M(Dex) macrophage activation, CCL2 was quantified in dialysates, gene expression ratios were determined from excised tissue surrounding the implant, histological analyses, and immunohistochemical analyses (CD68, CD163) were performed. Delayed Dex infusion resulted in the up-regulation of IL-6 at the transcript level in the tissue in contact with the microdialysis probe and decreased CCL2 concentrations collected in dialysates. Histological analyses showed increased cellular density as compared to controls in response to delayed Dex infusion. Dex delayed infusion resulted in an increased percentage of CD68(+)CD163(+), M(Dex), macrophages in the tissue surrounding the microdialysis probe as compared to probes that served as controls.

Expression of an immunoglobulin heavy chain transgene in macrophage as well as lymphocyte lineages in vivo.

A rearranged immunoglobulin heavy chain (IgH) transgene‐encoded protein is expressed in macrophage lineage cells, in addition to B and T lineages, in transgenic mouse bone marrow. Peripheral macrophages also express transgenic IgH protein. Mature T cells express lower levels than immature thymocytes. Almost all B220+ cells in the bone marrow express transgenic IgH protein, and this early expression in the B lineage is accompanied by a reduction of cell frequency even in the early B220+ CD43+ BP‐1− stages, although it is more prominent in BP‐1+ pre‐B cells. Thus, an IgH transgene can be expressed not only in lymphoid but also in myeloid cells, although its developmental effects are restricted to the B cell lineage.