Continuous CD8⁺ T-cell priming by dendritic cell cross-presentation of persistent antigen following adeno-associated virus-mediated gene delivery

Recombinant adeno-associated virus (rAAV) vectors establish persistent transgene expression in the skeletal muscle of mice. How dendritic cells acquire encoded antigens for CD8(+) T-cell priming is unknown. Here we document CD8(+) T-cell priming after lethal irradiation and bone marrow reconstitution of mice treated with an AAV vector several weeks earlier. Temporal separation of vector delivery and successful class I antigen presentation indicated that T-cell priming does not necessarily require antigen synthesis in AAV-transduced dendritic cells. An apparent cross-presentation of antigen acquired from muscle suggests that strategies to limit transgene expression in dendritic cells will not prevent unwanted CD8(+) T-cell responses.     

Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.     

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.     

TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.     

CD90-positive cells, an additional cell population, produce laminin alpha2 upon transplantation to dy(3k)/dy(3k) mice

Laminin alpha2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin alpha2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin alpha2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin alpha2 in non-myogenic cells of normal mice, and we could enrich these laminin alpha2-producing cells in CD90(+) cell fractions. Intriguingly, the number of CD90(+) cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin alpha2 in CD90(+) cells was not dependent on fusion with myogenic cells. Thus, CD90(+) cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.     

Immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4(+) T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8(+) T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8(+) tolerance in SM-Ova mice. We show herein that Ova-specific CD8(+) T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8(+) T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8(+) T cells.     

CD64 expression distinguishes monocyte-derived and conventional dendritic cells and reveals their distinct role during intramuscular immunization

Although most vaccines are administered i.m., little is known about the dendritic cells (DCs) that are present within skeletal muscles. In this article, we show that expression of CD64, the high-affinity IgG receptor FcγRI, distinguishes conventional DCs from monocyte-derived DCs (Mo-DCs). By using such a discriminatory marker, we defined the distinct DC subsets that reside in skeletal muscles and identified their migratory counterparts in draining lymph nodes (LNs). We further used this capability to analyze the functional specialization that exists among muscle DCs. After i.m. administration of Ag adsorbed to alum, we showed that alum-injected muscles contained large numbers of conventional DCs that belong to the CD8α(+)- and CD11b(+)-type DCs. Both conventional DC types were capable of capturing Ag and of migrating to draining LNs, where they efficiently activated naive T cells. In alum-injected muscles, Mo-DCs were as numerous as conventional DCs, but only a small fraction migrated to draining LNs. Therefore, alum by itself poorly induces Mo-DCs to migrate to draining LNs. We showed that addition of small amounts of LPS to alum enhanced Mo-DC migration. Considering that migratory Mo-DCs had, on a per cell basis, a higher capacity to induce IFN-γ-producing T cells than conventional DCs, the addition of LPS to alum enhanced the overall immunogenicity of Ags presented by muscle-derived DCs. Therefore, a full understanding of the role of adjuvants during i.m. vaccination needs to take into account the heterogeneous migratory and functional behavior of muscle DCs and Mo-DCs revealed in this study.     

CD8+ alpha beta+ T cells that lack surface CD5 antigen expression are a major lymphotactin (XCL1) source in peripheral blood lymphocytes

To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.     

Cutting edge: control of CD8+ T cell activation by CD4+CD25+ immunoregulatory cells

CD4(+)CD25(+) regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4(+)CD25(-) T cells and are potent suppressors of CD4(+)CD25(-) T cell activation in vitro. We demonstrate that CD4(+)CD25(+) T cells also suppress both proliferation and IFN-gamma production by CD8(+) T cells induced either by polyclonal or Ag-specific stimuli. CD4(+)CD25(+) T cells inhibit the activation of CD8(+) responders by inhibiting both IL-2 production and up-regulation of IL-2Ralpha-chain (CD25) expression. Suppression is mediated via a T-T interaction as activated CD4(+)CD25(+) T cells suppress the responses of TCR-transgenic CD8(+) T cells stimulated with soluble peptide-MHC class I tetramers in the complete absence of APC. These results broaden the immunoregulatory role played by CD4(+)CD25(+) T cells in the prevention of autoimmune diseases, but also raise the possibility that they may hinder the induction of effector CD8(+) T cells to tumor or foreign Ags.     

Increased shelterin mRNA expression in peripheral blood mononuclear cells and skeletal muscle following an ultra-long-distance running event

Located at the end of chromosomes, telomeres are progressively shortened with each replication of DNA during aging. Integral to the regulation of telomere length is a group of proteins making up the shelterin complex, whose tissue-specific function during physiological stress is not well understood. In this study, we examine the mRNA and protein levels of proteins within and associated with the shelterin complex in subjects (n = 8, mean age = 44 yr) who completed a physiological stress of seven marathons in 7 days. Twenty-two to 24 h after the last marathon, subjects had increased mRNA levels of DNA repair enzymes Ku70 and Ku80 (P < 0.05) in both skeletal muscle and peripheral blood mononuclear cells (PBMCs). Additionally, the PBMCs displayed an increment in three shelterin protein mRNA levels (TRF1, TRF2, and Pot-1, P < 0.05) following the event. Seven days of ultrarunning did not result in changes in mean telomere length, telomerase activity, hTert mRNA, or hterc mRNAs found in PBMCs. Higher protein concentrations of TRF2 were found in skeletal muscle vs. PBMCs at rest. Mean telomere length in skeletal muscle did not change and did not contain detectable levels of htert mRNA or telomerase activity. Furthermore, changes in the PBMCs could not be attributed to changes in the proportion of subtypes of CD4(+) or CD8(+) cells. We have provided the first evidence that, in humans, proteins within and associated with the shelterin complex increase at the mRNA level in response to a physiological stress differentially in PBMCs and skeletal muscle.     

Age-associated disruption of molecular clock expression in skeletal muscle of the spontaneously hypertensive rat

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.     

GITR expression on T-cell receptor-stimulated human CD8 T cell in a JNK-dependent pathway

Glucocorticoid-induced tumor necrosis factor receptor (TNFR) (GITR) family-related gene is a member of the TNFR super family. GITR works as one of the immunoregulatory molecule on CD4(+) regulatory T cells and has an important role on cell survival or cell death in CD4(+) T cells. Little is known about the expression of GITR on human CD8(+) T cells on antigen-specific and non-specific activation. Here, we report that expression of GITR on human CD8(+) T cells on T-cell receptor (TCR) (anti-CD3)-mediated stimulation is dependent on the JNK pathway. The activation of CD8(+) T cells was measured by the expression of IL-2 receptor-α (CD25), GITR and by IFN-γ production upon re-stimulation with anti-CD3 antibody. We studied the signaling pathway of such inducible expression of GITR on CD8(+) T cells. We found that a known JNK-specific inhibitor, SP600125, significantly down-regulates GITR expression on anti-CD3 antibody-mediated activated CD8(+) T cells by limiting JNK phosphorylation. Subsequently, after stimulation of the CD8(+) cells, we tested for the production of IFN-γ by the activated cells following restimulation with the same stimulus. It appears that the expression of GITR on activated human CD8(+) T cells might also be regulated through the JNK pathway when the activation is through TCR stimulation. Therefore, GITR serves as an activation marker on activated CD8(+) cells and interference with JNK phosphorylation, partially or completely, by varying the doses of SP600125 might have implications in CD8(+) cytotoxic T cell response in translational research.     

Mouse TCRalphabeta+CD8alphaalpha intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses

Mouse small intestine intraepithelial lymphocytes (IEL) that express alphabetaTCR and CD8alphaalpha homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined. To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis combined with real-time quantitative PCR and flow cytometry. Using these methods, TCRalphabeta(+)CD8alphaalpha IEL were compared with their TCRalphabeta(+)CD8beta(+) and TCRgammadelta(+) counterparts. Interestingly, TCRalphabeta(+)CD8alphaalpha IEL were found to preferentially express genes that would be expected to down-modulate their reactivity. They have a unique expression pattern of members of the Ly49 family of NK receptors and tend to express inhibitory receptors, along with some activating receptors. The signaling machinery of both TCRalphabeta(+)CD8alphaalpha and TCRgammadelta(+) IEL is constructed differently than other IEL and peripheral T cells, as evidenced by their low-level expression of the linker for activation of T cells and high expression of the non-T cell activation linker, which suppresses T cell activation. The TCRalphabeta(+)CD8alphaalpha IEL subset also has increased expression of genes that could be involved in immune regulation, including TGF-beta(3) and lymphocyte activation gene-3. Collectively, these data underscore the fact that, while TCRalphabeta(+)CD8alphaalpha IEL resemble TCRgammadelta(+) IEL, they are a unique population of cells with regulated Ag reactivity that could have regulatory function.     

A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways

Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80(+)CD11b(+)CD11c(+) bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80(+)CD11b(+)CD11c(-) bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80(+)CD11b(+)CD11c(+) cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80(+)CD11b(+)CD11c(+) cells express increased inflammatory markers compared with F4/80(+)CD11b(+)CD11c(-) cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c(+) myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance.