Lorna B. Jarvis

Autoreactive human peripheral blood CD8+ T cells with a regulatory phenotype and function

Despite substantial advances in our understanding of CD4+CD25+ regulatory T cells, a possible equivalent regulatory subset within the CD8+ T cell population has received less attention. We now describe novel human CD8+/TCRαβ+ T cells that have a regulatory phenotype and function. We expanded and cloned these cells using autologous LPS‐activated dendritic cells. The clones were not cytolytic, but responded in an autoreactive HLA class I‐restricted fashion, by proliferation and production of IL‐4, IL‐5, IL‐13 and TGFβ1, but not IFN‐γ. They constitutively expressed CD69 and CD25 as well as molecules associated with CD4+CD25+ regulatory T cells, including cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) and Foxp3. They suppressed IFN‐γ production and proliferation by CD4+ T cells in vitro in a cell contact‐dependent manner, which could be blocked using a CTLA‐4‐specific mAb. They were more readily isolated from patients with ankylosing spondylitis and may therefore be up‐regulated in response to inflammation. We suggest that they are the CD8+ counterparts of CD4+CD25+ regulatory T cells. They resemble recently described CD8+ regulatory cells in the rat that were able to abrogate graft‐versus‐host disease. Likewise, human HLA‐restricted CD8+ regulatory T cells that can be cloned and expanded in vitro may have therapeutic applications.

CD27 expression discriminates between regulatory and non-regulatory cells after expansion of human peripheral blood CD4+ CD25+ cells

It is clear that regulatory T cells (Treg) have an important role in preventing autoimmunity and modulating responses to pathogens. Full characterization of Treg cell function in human patients would be greatly facilitated by practical methods for expanding Treg in vitro. Methods for expansion have been reported but whether expression of surface and intracellular markers associated with freshly isolated Treg following expansion correlates with the maintenance of function is unclear. Our aim was to investigate the various methods of expansion and to correlate regulatory activity with expression of these markers. We show that, of the markers associated with freshly isolated Treg, only CD27 expression correlated with regulatory activity and could be used to isolate cells with regulatory activity from lines expanded from CD4+ CD25+ cells. Also, cells expressing high levels of the transcription factor forkhead box P3 (Foxp3) were confined to the CD27+ population within these lines. Expression of CD27 by cells in lines expanded from CD4+ CD25– cells varied depending on the stimulus used for expansion, but these lines did not have significant regulatory activity even when the CD27+ cells were tested. Analysis of synovial CD4+ CD25+ cells from reactive arthritis patients revealed that they were predominantly CD27 positive. This also applied to CD25high and CD25intermediate CD4+ cells, despite their reported different abilities to regulate. We conclude that, whilst CD27 is useful for identifying Treg in the cell lines obtained after expansion of CD4+ CD25+ cells, its expression may not reliably identify the Treg cell population in other T‐cell populations such as those found in joints.

Magnetic resonance imaging with hyperpolarized [1,4-(13)C2]fumarate allows detection of early renal acute tubular necrosis.

Acute kidney injury (AKI) is a common and important medical problem, affecting 10% of hospitalized patients, and it is associated with significant morbidity and mortality. The most frequent cause of AKI is acute tubular necrosis (ATN). Current imaging techniques and biomarkers do not allow ATN to be reliably differentiated from important differential diagnoses, such as acute glomerulonephritis (GN). We investigated whether 13C magnetic resonance spectroscopic imaging (MRSI) might allow the noninvasive diagnosis of ATN. 13C MRSI of hyperpolarized [1,4-13C2]fumarate and pyruvate was used in murine models of ATN and acute GN (NZM2410 mice with lupus nephritis). A significant increase in [1,4-13C2]malate signal was identified in the kidneys of mice with ATN early in the disease course before the onset of severe histological changes. No such increase in renal [1,4-13C2]malate was observed in mice with acute GN. The kidney [1-13C]pyruvate/[1-13C]lactate ratio showed substantial variability and was not significantly decreased in animals with ATN or increased in animals with GN. In conclusion, MRSI of hyperpolarized [1,4-13C2]fumarate allows the detection of early tubular necrosis and its distinction from glomerular inflammation in murine models. This technique may have the potential to identify a window of therapeutic opportunity in which emerging therapies might be applied to patients with ATN, reducing the need for acute dialysis with its attendant morbidity and cost.

MiR-210 Is Induced by Oct-2, Regulates B Cells, and Inhibits Autoantibody Production

MicroRNAs (MiRs) are small, noncoding RNAs that regulate gene expression posttranscriptionally. In this study, we show that MiR-210 is induced by Oct-2, a key transcriptional mediator of B cell activation. Germline deletion of MiR-210 results in the development of autoantibodies from 5 mo of age. Overexpression of MiR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice overexpressing MiR-210 exhibited impaired class-switched Ab responses, a finding confirmed in wild-type B cells transfected with a MiR-210 mimic. In vitro studies demonstrated defects in cellular proliferation and cell cycle entry, which were consistent with the transcriptomic analysis demonstrating downregulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of MiR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.

Regulatory IL4+CD8+ T cells in patients with ankylosing spondylitis and healthy controls

Objectives: Interleukin (IL)4+CD8+ regulatory T cells (Treg) obtained from peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis (AS) by coculture with autologous dendritic cells (DC) have been previously described. In the present work, the proportions of IL4+CD8+ T cells in PB from patients with AS and controls are examined; in addition the conditions required for the generation of IL4+CD8+ Treg cells and their frequency in T cell lines from patients with AS and controls are investigated. Methods: CD8+ T cells were either stimulated non-specifically ex vivo and intracellular cytokines examined, or cocultured with DC and other stimuli, for 2 weeks. The resulting lines were analysed for cytokine expression. Clones derived from these lines were tested for regulatory function. Results: PBMCs from patients with AS and from human leukocyte antigen (HLA)-B27+ healthy controls contained a higher frequency of IL4+CD8+ T cells than those from HLA-B27− controls. Likewise, CD8+ T cell lines obtained by coculture with DC contained a higher ratio of IL4+ to interferon (IFN)γ+ cells when obtained from patients with AS or HLA-B27+ controls. T cell clones obtained from these lines showed regulatory activity. Outgrowth of IL4+ CD8+ T cells required contact with DC, but not maturation with lipopolysaccharide (LPS); allogeneic DC were also effective. Coculture with lymphoblastoid cells, or anti-CD3/CD28 microbeads, produced only expansion of IFNγ-producing CD8+ T cells. Conclusions: The higher proportion of CD8+ cells which can produce IL4 in PB and in expanded CD8+ T cell lines suggests an altered pattern of CD8+ T cell differentiation in AS and in HLA-B27+ healthy individuals. This predisposition to generate IL4+CD8+ T cells may play a role in pathogenesis of spondyloarthritis.

Increased IL-4+ CD8+ T cells in peripheral blood and autoreactive CD8+ T cell lines of patients with inflammatory arthritis

OBJECTIVE To measure the frequencies of IL-4+ CD8+ T cells from patients with AS and RA, and to assess their clinical relevance and properties. METHODS Peripheral blood (PB) and clinical data were obtained from 37 AS, 36 RA patients and 37 healthy controls. We also generated IL-4-producing CD8+ T cell lines and clones by co-culture with autologous dendritic cells. Using flow cytometry, we evaluated intracellular cytokine expression by T cells following stimulation with PMA and calcium ionophore. The phenotype and ability of the IL-4-producing CD8+ T cell clones to suppress IFN-gamma production were examined. RESULTS The percentages of IL-4+ CD8+ T cells were higher in PB of patients with AS and RA than controls (medians 0.90 and 0.84% vs 0.30%). In RA, patients with active inflammation had an increased percentage of IL-4+ CD8+ T cells. Higher frequencies of IL-4+ CD8+ T cells were also found in CD8+ T cell lines established from patients with arthritis. Interestingly, most IL-4+ CD8+ T cells produced TNF-alpha. Cloning the CD8+ T cell lines yielded more IL-4-producing clones from AS (23%) and RA patients (14%) than from controls (7%). The ability to suppress IFN-gamma production was observed in 56% (AS) and 85% (RA) of IL-4-producing clones. Suppressive IL-4+ CD8+ T cell clones from RA patients showed a similar regulatory phenotype to the clones previously isolated from AS patients. CONCLUSIONS Expansion of IL-4+ CD8+ T cells, which may include precursors of a regulatory CD8+ T cell subset, may represent a general response to chronic joint inflammation.

Extracellular Lactate: A Novel Measure of T Cell Proliferation.

Following activation, T cells rapidly divide and acquire effector functions. This energetically demanding process depends upon the ability of T cells to undergo metabolic remodeling from oxidative phosphorylation to aerobic glycolysis, during which glucose is converted into lactate and released extracellularly. In this article, we demonstrate that extracellular lactate can be used to dynamically assess human T cell responses in vitro. Extracellular lactate levels strongly correlated with T cell proliferation, and measuring lactate compared favorably with traditional methods for determining T cell responses (i.e., [3H]thymidine incorporation and the use of cell proliferation dyes). Furthermore, we demonstrate the usefulness of measuring lactate as a read-out in conventional suppression assays and high-throughput peptide-screening assays. Extracellular lactate was stably produced over 7 d, and results were reproducibly performed over several freeze–thaw cycles. We conclude that the use of extracellular lactate measurements can be a sensitive, safe, stable, and easy-to-implement research tool for measuring T cell responses and cellular metabolic changes in vitro.