IL-12 and IL-18 induce interferon-γ production and de novo CD2 expression in porcine γδ T cells

γδ T cells are highly abundant in the blood and spleen of pigs but little is known about their functional differentiation. In this study the potential of the type-1 polarizing cytokines IL-12 and IL-18 in combination with IL-2 and Concanavalin A (ConA) to stimulate porcine γδ T cells was investigated. Stimulation of purified γδ T cells with ConA and IL-2 induced a strong proliferation of CD2⁻ γδ T cells, whereas additional stimulation with IL-12 and IL-18 caused a stronger proliferation of CD2⁺ γδ T cells. IFN-γ could only be detected in supernatants of γδ T-cell cultures supplemented with IL-12 and IL-18. Experiments with sorted CD2/SWC5-defined γδ T-cell subsets revealed that CD2⁺SWC5⁻ γδ T cells are the main producers of IFN-γ following stimulation with IL-2/IL-12/IL-18. Additional stimulation with ConA led to an upregulation of CD2 within the CD2⁻ γδ T cell subsets, indicating a previously unnoticed plasticity of CD2-defined γδ T cell subsets.     

Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease

Gut intraepithelial γδ and CD8⁺ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38⁺), gut-homing (CD103⁺) γδ and CD8⁺ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38⁺CD103⁺) γδ and CD8⁺ αβ T cells, respectively, while 16 had increase in gluten-specific CD4⁺ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8⁺ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8⁺ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4⁺ T cells appear to drive expansion of clonally diverse γδ or CD8⁺ αβ T-cell clonotypes that may not be specific for the gluten antigen.     

NKG2D receptor activation of NF-κB enhances inflammatory cytokine production in murine effector CD8 T cells

To induce strong immune responses, naïve CD8⁺ T cells require stimulation through the TCR and costimulatory receptors. However, the biological effect of activating costimulatory receptors on effector T cells is still unclear. One costimulatory receptor that is likely to be engaged at the target site is NKG2D. This activating receptor is expressed on human and murine CD8⁺ T cells with its ligands expressed on the majority of tumor cells and during some infections. In order to determine how activation of costimulatory receptors alters effector CD8⁺ T cell functions, this study compared the activation of the NF-κB signaling pathway by two costimulatory receptors, CD28 and NKG2D. Compared to CD28 costimulation, activation of murine effector CD8⁺ T cells through CD3 and NKG2D receptors enhanced activation of NF-κB as shown by increased phosphorylation of IKKα, IκBα, and NF-κB and IκBα degradation. NKG2D costimulation also increased activation, nuclear translocation, and DNA binding of NF-κB p65/p50 dimers. Activation of the NF-κB pathway also lead to increased gene expression and secretion of pro-inflammatory cytokines, including IFNα and IFNγ, and decreased gene expression and secretion of anti-inflammatory cytokines, including IL-10 and CCL2. Altered NF-κB activation also increased expression of the effector molecules TNFα, lymphotoxins α and β, and Fas ligand, and increased tumor cell killing through FasL. These data show that compared to CD28 costimulation, activation through the NKG2D receptor leads to the differential activation of the NF-κB signaling pathway and potentially enhances the anti-tumor and anti-viral functions of effector CD8⁺ T cells.     

Two functionally distinct subsets of IL-17 producing γδ T cells

The γδ T cells play an important role in both mice and humans as a source of the cytokine IL-17, which is key for immune resistance to certain pathogens. In mice, most of these IL-17 producers, termed γδT-17 cells, actually comprise two distinct types: those expressing an invariant Vγ6Vδ1⁺ TCR and those expressing a Vγ4⁺ TCR. Murine γδT-17 cells acquire an inherent bias to produce IL-17 and other “type 17” cytokines during thymic development. The similarities and differences between the two mouse γδT-17 types are reviewed here, and the potential implications of their differences are discussed. There is some evidence that two distinct TCR-defined IL-17-producing γδ T cell subsets also exist in humans, but unlike the mouse γδT-17 cells, these cells are probably not imprinted with an IL-17 bias during thymic development, but rather acquire an IL-17 bias in the periphery.     

Acute oxidative stress affects IL-8 and TNF-α expression in IPEC-J2 porcine epithelial cells

Reactive oxygen species are implicated in cell and tissue damage in a number of diseases including acute and chronic inflammation of the gut. Effects of H₂O₂ exposure on non-carcinogenic porcine epithelial cell line, IPEC-J2 cells cultured on collagen-coated membrane inserts were monitored based on transepithelial electrical resistance (TER) change, extent of necrotic cell damage, gene expression of inflammatory cytokines IL-8 and TNF-α. Furthermore, the junction proteins claudin-1 and E-cadherin were also investigated by immunohistochemistry. Peroxide (1mM) increased IL-8 and TNF-α gene expression levels significantly allowing 1 h recovery time without affecting the cellular distribution of junction proteins, TER and cell survival rate. In conclusion, the IPEC-J2 cell line on membrane insert was introduced as a fast and reliable investigation tool for oxidative stimuli-triggered intestinal inflammation and in the future as a screening method for antioxidant and probiotic candidates.     

The promise of γδ T cells and the γδ T cell receptor for cancer immunotherapy

γδ T cells form an important part of adaptive immune responses against infections and malignant transformation. The molecular targets of human γδ T cell receptors (TCRs) remain largely unknown, but recent studies have confirmed the recognition of phosphorylated prenyl metabolites, lipids in complex with CD1 molecules and markers of cellular stress. All of these molecules are upregulated on various cancer types, highlighting the potential importance of the γδ T cell compartment in cancer immunosurveillance and paving the way for the use of γδ TCRs in cancer therapy. Ligand recognition by the γδ TCR often requires accessory/co-stimulatory stress molecules on both T cells and target cells; this cellular stress context therefore provides a failsafe against harmful self-reactivity. Unlike αβ T cells, γδ T cells recognise their targets irrespective of HLA haplotype and therefore offer exciting possibilities for off-the-shelf, pan-population cancer immunotherapies. Here, we present a review of known ligands of human γδ T cells and discuss the promise of harnessing these cells for cancer treatment.     

Regulation of Con A – dependent cytokine production from CD4+ and CD8+ T lymphocytes by autosecretion of histamine

Objectives: Previously we have shown that both CD4+ T cells and CD8+ T cells produce histamine when activated with Con A. The aim of this study was to examine whether cytokine production by these cells is regulated by autosecretion of histamine.Materials: CD4+ and CD8+ T cells were separated from spleen cells of C57BL/6 mice and mice lacking the H1 receptor (H1R) or H2R, using anti – CD4+ – and anti – CD8+ – coupled magnetic beads, respectively.Results: Depletion of the H1R resulted in decreases in the release of IL-2 and IL-10 from both CD4+ and CD8+ cells and increases in the release of IL-4 from CD4+ T cells and IFN- from CD8+ cells. Mice lacking the H2R showed up – regulation of IFN- secretion from CD8+ cells and of IL-4 from CD4+ and CD8+ T cells. Release of IL-2 and IL-10 from CD4+ as well as CD8+ cells was down – regulated in these mice. Both CD4+ and CD8+ T cell fractions synthesized histamine, which was enhanced in the H1R - deficient CD8+ T cells. Treatment of the cells with -fluoromethyl-histidine, a specific inhibitor of HDC, or histaminase increased IFN- from CD8+ cells, whereas it had no appreciable effect on IL-4 secretion from CD4+ cells.Conclusions: These results suggest that cytokine production by CD4+ and CD8+ T lymphocytes is regulated by autosecretion of histamine.Received 4 July 2003; returned for revision 23 September 2003; returned for final revision 13 October 2003, accepted by M. Parnham 17 October 2003     

Are CD8+CD122+ cells regulatory T cells or memory T cells?

We identified CD8(+)CD122(+) regulatory T cells in the mouse. Some immunologists consider CD8(+)CD122(+) cells to be memory T cells despite our report of their regulatory function. Here, we propose a dual phenotype of these cells. Murine CD8(+)CD122(+) T cells demonstrate both memory and regulatory features in their functional profiles. Human CD8(+)CXCR3(+) T cells, which are thought to be the human counterpart of murine CD8(+)CD122(+) regulatory T cells, do not match human central memory T cells of the CD8(+)CD45RA(-)CCR7(+) phenotype. Thus, we must consider human CD8(+) regulatory T cells and murine CD8(+) regulatory T cells separately. Of human CD8(+) regulatory T cells, CD8(+)CXCR3(+) regulatory T cells can be divided into further subsets and we may be able to distinguish memory T cells and regulatory T cells. Of murine CD8(+)CD122(+) regulatory T cells, it seems to be impossible to divide CD8(+)CD122(+)CD44(+)CD62L(+) regulatory T cells into further subsets at present, indicating that this single population of cells has activities of both regulatory T cells and memory T cells.     

β2-Microglobulin-free HLA-G activates natural killer cells by increasing cytotoxicity and proinflammatory cytokine production

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule and plays a role in tissue specific immunoregulation. Many studies have addressed functional aspects of β2-microglobulin (β2m)-associated HLA-G1. β2m-free HLA-G has been found in human placental cytotrophoblasts and pancreatic β cells although its function remains unclear. In the present study, we investigated the function of β2m-free HLA-G by transfecting HLA-G1 and -G3 into human β2m deficient rat pancreatic β cell carcinoma (BRIN-BD11) cells. RT-PCR and western blots studies confirmed high expression of HLA-G1 and -G3 in -G1 and -G3 transfectants, respectively. HLA-G1 and -G3 were detected mainly in intracellular compartments of BRIN-BD11 transductants by confocal fluorescent microscopy and flow cytometry. Functional analysis revealed that β2m-free HLA-G promoted xenogeneic cytotoxic lysis of BRIN-BD11 cells by natural killer (NK) cells and increased production of IL-1β, TNF-α, and IFN-γ. Stimulation of cytotoxic lysis was impaired by blocking the MAPK and DNA-PKcs pathways in NK cells. Importantly, treatment with 33mAb, a KLR2DL4 receptor agonist, induced NK-mediated cytotoxic lysis of BRIN-BD11 cells transfected with a mock vector. Our data suggest that β2m-free HLA-G activates NK cells via engagement of KLR2DL4 receptors.     

Mucosal-associated invariant T cells and Vδ2+ γδ T cells in community acquired pneumonia: association of abundance in sputum with clinical severity and outcome

Mucosal-associated invariant T (MAIT) cells and Vδ2⁺ γδ T cells are anti-bacterial innate-like lymphocytes (ILLs) that are enriched in blood and mucosa. ILLs have been implicated in control of infection. However, the role of ILLs in community-acquired pneumonia (CAP) is unknown. Using sputum samples from a well-characterized CAP cohort, MAIT cell and Vδ2⁺ T cell abundance was determined by quantitative polymerase chain reaction (qPCR). Cytokine and chemokine concentrations in sputum were measured. The capacity of bacteria in sputum to produce activating ligands for MAIT cells and Vδ2⁺ T cells was inferred by 16S rRNA sequencing. MAIT cell abundance in sputum was higher in patients with less severe pneumonia; duration of hospital admission was inversely correlated with both MAIT and Vδ2⁺ T cell abundance. The abundance of both ILLs was higher in patients with a confirmed bacterial aetiology; however, there was no correlation with total bacterial load or the predicted capacity of bacteria to produce activating ligands. Sputum MAIT cell abundance was associated with interferon (IFN)-α, IFN-γ, and sputum neutrophil abundance, while Vδ2⁺ T cell abundance was associated with CXCL11 and IFN-γ. Therefore, MAIT and Vδ2⁺ T cells can be detected in sputum in CAP, where they may contribute to improved clinical outcome.     

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8⁺ T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103⁺ CD11b⁻ CD8α⁺ DCs cross-present IEC-derived ovalbumin to CD8⁺ OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c⁺ MHCII^hi CD8α⁺ migratory DCs, but absent from all other subsets, including the resident CD8α^hi DCs. Crucially, delivery of purified CD8α⁺ LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8⁺ T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α⁺ LDCs were uniquely able to cross-prime interferon γ-producing CD8⁺ T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α⁺ intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8⁺ T cells. They may therefore represent an important target for the development of antiviral vaccinations.     

Mechanisms underlying lineage commitment and plasticity of human γδ T cells

Phenotypic and functional heterogeneity are the hallmarks of effector and memory T cells. Upon antigen stimulation, γδ T cells differentiate into two major types of memory T cells: central memory cells, which patrol the blood and secondary lymphoid organs, and effector memory cells, which migrate to peripheral tissues. γδ T cells display in vitro a certain degree of plasticity in their function that is reminiscent of that which is observed in conventional CD4 T cells. Similar to CD4 T cells, in which a plethora of specialized subsets affect the host response, γδ T cells may readily and rapidly assume distinct Th1-, Th2-, Th17-, T_FH and T regulatory-like effector functions, suggesting that they profoundly influence cell-mediated and humoral immune responses. In addition to differences in cytokine repertoire, γδ T cells exhibit diversity in homing, such as migration to lymph node follicles, to help B cells versus migration to inflamed tissues. Here, we review our current understanding of γδ T-cell lineage heterogeneity and flexibility, with an emphasis on the human system, and propose a classification of effector γδ T cells based on distinct functional phenotypes.