Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation.

Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

Characterization of NKR+ T-cell subsets in human bone marrow:implications for immunosurveillance of neoplasia

In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.

Microbial Recognition Via Toll-Like Receptor-Dependent and -Independent Pathways Determines the Cytokine Response of Murine Dendritic Cell Subsets to CD40 Triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

Relationships Among Murine CD11chigh 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.

Tracking phenotypically and functionally distinct T cell subsets via T cell repertoire diversity

Antigen-specific T cell receptors (TCRs) recognise complexes of immunogenic peptides (p) and major histocompatibility complex (MHC) glycoproteins. Responding T cell populations show profiles of preferred usage (or bias) toward one or few TCR&beta; chains. Such skewing is also observed, though less commonly, in TCRα chain usage. The extent and character of clonal diversity within individual, antigen-specific T cell sets can be established by sequence analysis of the TCRV&beta; and/or TCRVα CDR3 loops. The present review provides examples of such TCR repertoires in prominent responses to acute and persistent viruses. The determining role of structural constraints and antigen dose is discussed, as is the way that functionally and phenotypically distinct populations can be defined at the clonal level. In addition, clonal dissection of “high” versus “low” avidity, or “central” versus “effector” memory sets provides insights into how these antigen specific T cell responses are generated and maintained. As TCR diversity potentially influences both the protective capacity of CD8+ T cells and the subversion of immune control that leads to viral escape, analysing the spectrum of TCR selection and maintenance has implications for improving the functional efficacy of T cell responsiveness and effector function.

The ontogeny of naïve and regulatory CD4(+) T-cell subsets during the first postnatal year: a cohort study

As there is limited knowledge regarding the longitudinal development and early ontogeny of naïve and regulatory CD4(+) T-cell subsets during the first postnatal year, we sought to evaluate the changes in proportion of naïve (thymic and central) and regulatory (resting and activated) CD4(+) T-cell populations during the first postnatal year. Blood samples were collected and analyzed at birth, 6 and 12 months of age from a population-derived sample of 130 infants. The proportion of naïve and regulatory CD4(+) T-cell populations was determined by flow cytometry, and the thymic and central naïve populations were sorted and their phenotype confirmed by relative expression of T cell-receptor excision circle DNA (TREC). At birth, the majority (94%) of CD4(+) T cells were naïve (CD45RA(+)), and of these, ~80% had a thymic naïve phenotype (CD31(+) and high TREC), with the remainder already central naïve cells (CD31(-) and low TREC). During the first year of life, the naïve CD4(+) T cells retained an overall thymic phenotype but decreased steadily. From birth to 6 months of age, the proportion of both resting naïve T regulatory cells (rTreg; CD4(+)CD45RA(+)FoxP3(+)) and activated Treg (aTreg, CD4(+)CD45RA(-)FoxP3(high)) increased markedly. The ratio of thymic to central naïve CD4(+) T cells was lower in males throughout the first postnatal year indicating early sexual dimorphism in immune development. This longitudinal study defines proportions of CD4(+) T-cell populations during the first year of postnatal life that provide a better understanding of normal immune development.

Telomere length in human natural killer cell subsets

Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. In the elderly, the cytotoxic function of NK cells is often compromised. Telomeres progressively shorten with each cell division and with age in most somatic cells eventually leading to chromosomal instability and cellular senescence. We studied the telomere length in NK cell subsets isolated from peripheral blood using "flow FISH," a method in which the hybridization of telomere probe in cells of interest is measured relative to internal controls in the same tube. We found that the average telomere length in human NK cells decreased with age as was previously found for human T lymphocytes. Separation of adult NK cells based on CD56 and CD16 expression revealed that the telomere length was significantly shorter in CD56(dim)CD16(+) (mature) NK cells compared to CD56(bright)CD16(-) (immature) NK cells from the same donor. Furthermore, sorting of NK cells based on expression of activation markers, such as NKG2D and LFA-1, revealed that NK cells expressing these markers have significantly shorter telomeres. Telomere fluorescence was very heterogeneous in NK cells expressing CD94, killer inhibitory receptor (KIR), NKG2A, or CD161. Our observations indicate that telomeric DNA in NK cells is lost with cell division and with age similar to what has been observed for most other hematopoietic cells. Telomere attrition in NK cells is a plausible cause for diminished NK cell function in the elderly.

Beta1 integrins differentially control extravasation of inflammatory cell subsets into the CNS during autoimmunity

Inhibiting the alpha(4) subunit of the integrin heterodimers alpha(4)beta(1) and alpha(4)beta(7) with the monoclonal antibody natalizumab is an effective treatment for multiple sclerosis (MS). However, the pharmacological action of natalizumab is not understood conclusively. Previous studies suggested that natalizumab inhibits activation, proliferation, or extravasation of inflammatory cells. To specify which mechanisms, cell types, and alpha(4) heterodimers are affected by the antibody treatment, we studied MS-like experimental autoimmune encephalomyelitis (EAE) in mice lacking the beta(1)-integrin gene either in all hematopoietic cells or selectively in T lymphocytes. Our results show that T cells critically rely on beta(1) integrins to accumulate in the central nervous system (CNS) during EAE, whereas CNS infiltration of beta(1)-deficient myeloid cells remains unaffected, suggesting that T cells are the main target of anti-alpha(4)-antibody blockade. We demonstrate that beta(1)-integrin expression on encephalitogenic T cells is critical for EAE development, and we therefore exclude alpha(4)beta(7) as a target integrin of the antibody treatment. T cells lacking beta(1) integrin are unable to firmly adhere to CNS endothelium in vivo, whereas their priming and expansion remain unaffected. Collectively, these results suggest that the primary action of natalizumab is interference with T cell extravasation via inhibition of alpha(4)beta(1) integrins.