CD4+ and γδTCR+ T lymphocytes are sources of interleukin-17 in swine

In the veterinary field, only limited information is available about interleukin-17A (IL-17), despite the fact that this cytokine plays an important role during pro-inflammatory immune responses and induces the production of chemotactic factors for neutrophils. The aim of this study was to characterize porcine IL-17-producing cells. We tested the cross-reactivity of five anti-human IL-17 monoclonal antibodies because such antibodies against porcine IL-17 are currently unavailable. Whole blood cells (WBCs) were stimulated with phorbol-myristate-acetate (PMA) and ionomycin and subsequently analyzed by flow cytometry. The antibody clone SCPL1362 was found to cross-react with porcine IL-17, whereas the other four antibodies tested did not recognize this cytokine. Using this antibody, we characterized porcine WBC-secreting IL-17 after PMA and ionomycin stimulation. All IL-17-producing WBCs were positive for the T lymphocyte marker CD3. Myeloid cells (CD172α(+)) and B lymphocytes (CD79α(+)) were IL-17 negative. The major subset of IL-17 positive T lymphocytes was the CD4(+) lymphocytes (about 60% of all IL-17 positive WBCs). The remaining IL-17 positive WBCs were γδTCR(+) lymphocytes. CD8 positive and CD8 negative cells were found within both CD4(+) and γδTCR(+) cells producing the cytokine. Moreover, IL-17 positive cells were mostly CD45RA negative, therefore activated cells or memory cells. Flow cytometry data were confirmed using sorted cells. Both sorted CD4(+) and γδTCR(+) cells produced IL-17 at mRNA level after PMA and ionomycin stimulation while double negative CD4(-)γδTCR(-) cells were negative for IL-17. We can conclude that only two subpopulations of porcine WBCs are sources of IL-17 after non-specific stimulation: CD3(+)CD4(+) and CD3(+)γδTCR(+).     

Folylpoly-γ-glutamate synthesis by bacteria and mammalian cells

Summary The purification and properties of folylpolyglutamate synthetase fromCorynebacterium sp, and some properties of partially purified enzyme fromLactobacillus casei, Streptococcus faecalis, Neurospora crassa, pig liver, and Chinese hamster ovary cells, are described.TheCorynebacterium enzyme catalyzes a MgATP-dependent addition of glutamate to a variety of reduced pteroate and pteroylmono-, di-, and triglutamate substrates, with the concomitant production of MgADP and phosphate. Although glutamate moieties are added in a sequential fashion, the kinetic mechanism, which is Ordered Ter Ter, precludes the sequential addition of glutamate moieties to enzyme-bound folate. It is suggested that catalysis precedes via the formation of a pteroyl--glutamyl phosphate intermediate.Thein vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differ from source to source, appear to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources.Only one enzyme appears to be involved in the conversion of pteroylmonoglutamates to polyglutamate forms in both bacteria and mammalian cells. Bacterial folylpolyglutamate synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroylpolyglutamates as their preferred, and sometimes only, polyglutamate substrate. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of mammalian folylpolyglutamate synthetases.     

α-Carbonic anhydrases are strongly activated by spinaceamine derivatives

A series of 4-substituted-spinaceamine (4,5,6,7-tetrahydro-imidazolo[4,5-c]pyridine) were prepared from histamine and aromatic aldehydes Schiff bases, and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, and the nature of the moiety in position 4 of the bicyclic system was the factor influencing activation properties against all isoforms. For hCA I, these compounds showed K_As in the range of 2.52–21.5?µM, the most effective activator being 4-(2-hydroxyphenyl)-spinaceamine. For hCA II the activation constants ranged between 0.60 and 17.2?µM, with 4-(2,3,5,6-tetrafluorophenyl)- spinaceamine the best activator. Affinity for hCA IV was in the range of 0.52–63.8?µM, and the same compound as for hCA II was the most effective activator. The most sensitive isoform for activation was the brain-associated hCA VII, for which K_As in the range of 82?nM–4.26?µM were observed. Effective hCA VII activators were the (2-bromophenyl)-, 2,3,5,6-tetrafluorophenyl- and furyl-substituted spineaceamines (K_As of 82–95?nM). As CA activators may have pharmacologic applications in various fields, this work provides interesting derivatives for further studies.     

Arthritis: where are the T cells?

T-helper (Th) lymphocytes contribute to arthritis pathogenesis by helping B cells to produce antibodies, by producing cytokines that activate effector cells involved in the destruction of cartilage and bone, and by contributing to osteoclast differentiation. There are murine models of arthritis, most notably collagen- and proteoglycan-induced arthritis, in which arthritis depends on T-cell recognition of antigens that are expressed in the joints. In spite of this, we still do not know the antigens recognised by arthritogenic Th cells in humans. Moreover, current evidence for Th cells exerting arthritogenic effector functions within the joints is only indirect.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

Cutting edge: Generation of colitogenic Th17 CD4 T cells is enhanced by IL-17+ γδ T cells

Th 17 cells have been implicated in the pathogenesis of colitis; however, a cellular mechanism by which colitogenic Th17 immunity arises in vivo remains unclear. In this study, we report that a subset of IL-17(+) γδ T cells plays a crucial role in enhancing in vivo Th17 differentiation and T cell-mediated colitis. TCRβ(-/-) mice were highly susceptible to T cell-mediated colitis, whereas TCRβδ(-/-) mice were resistant to the disease. Importantly, cotransfer of IL-17(+) but not of IL-17(-) γδ T cells with CD4 T cells was sufficient to enhance Th17 differentiation and induce full-blown colitis in TCRβδ(-/-) recipients. Collectively, our results provide a novel function of IL-17(+) γδ T cell subsets in supporting in vivo Th17 differentiation and possibly in fostering the development of intestinal inflammation.     

Unprimed CD4+ and CD8+ T cells can be rapidly activated by a CD3×CD19 bispecific antibody to proliferate and become cytotoxic

We previously reported that a CD3×CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3×CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3×CD19 bsAb. Within the same time spaancytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3×CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.This work was supported by grant IKMN 90-10 from the Dutch Cancer Society. M.C. was supported by a grant from the UK Medical Research Couneil     

Lung cancer patients’ CD4<Superscript>+</Superscript> T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients' T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4(+) T lymphocytes in the activation of cytotoxic CD8(+) T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4(+) T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4(+) T cells. METHODS: Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4(+) T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. RESULTS: The vaccines activated CD4(+) T cells from all ten patients, despite the presence of CD33(+)CD11b(+) MDSC. Activated CD4(+) T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. CONCLUSIONS: The NSCLC vaccines activate tumor-specific CD4(+) T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.     

In vitro sensitization with HSV-lnfected cells induces Tμ cells to become cytotoxic Tγ cells which are theophylline resistant

Following in vitro sensitization with HSV-infected cells, T_γ cells comprise most of the cytotoxic effector cell population. However, whereas freshly obtained T_γ cells exhibit theophylline sensitivity in the sheep erythrocyte rosette assay, presensitized T_γ cells are theophylline resistant. Similarly, when T cells are fractionated according to their theophylline sensitivity before the sensitization culture, theophylline-resistant T_μ cells appear as the precursors of T_γ cytotoxic effector cells, the T_μ-T_γ switch occurring with a transitory eclipse of Fc receptors, and maintenance of theophylline resistance.     

Large intestine intraepithelial lymphocytes from Apc+/+ and Apc+/Min mice and their modulation by indigestible carbohydrates: the IL-15/IL-15Rα complex and CD4+CD25+ T cells are the main targets

We have shown recently that some indigestible carbohydrate (short-chain fructo-oligosaccharides [sc-FOS]) reduced colon tumor incidence in Apc+/Min mice, and that this effect depended on a functional local immune system. In addition, IL-15 mRNA was concomitantly modulated in the mucosa. Since intraepithelial lymphocytes (IELs) are in close contact with intestinal epithelial cells, these cells are the candidates most likely to be involved in early cancer immunosurveillance. The present study documents the effects of sc-FOS on large intestine IELs (LI-IELs) from Apc+/+ or Apc+/Min mice by analyzing markers related to their phenotype, their activation status, and the cell surface IL-15/IL-15R. In the colons of Apc+/Min mice, fewer LI-IELs expressed surface IL-15/IL-15R. In addition, a lower number of CD4⁺ LI-IELs expressed CD25, although more LI-IELs expressed CD69, as compared to normal mice. The sc-FOS–enriched diet caused a decrease in the proportion of CD25⁺ LI-IELs and an increase in the percentage of LI-IELs bearing surface IL-15/IL-15R, independently of the Apc gene status. The IL-15/IL-15R increase was, however, higher in Min mice, and returned to a level very similar to that of Apc+/+ mice when the latter mice were fed a low-fiber diet. The sc-FOS-enriched diet specifically induced an increase in CD69⁺ cells in Apc+/+ mice, and a decrease in the proportion of CD4⁺CD25⁺ LI-IELs in Apc+/Min mice. Some of these modulations could contribute to the development of a better immune anticancer response in the early steps of cancer development.     

Why are large conformational changes well described by harmonic normal modes?

Low-frequency normal modes generated by elastic network models tend to correlate strongly with large conformational changes of proteins, despite their reliance on the harmonic approximation, which is only valid in close proximity of the native structure. We consider 12 variants of the torsional network model (TNM), an elastic network model in torsion angle space, that adopt different sets of torsion angles as degrees of freedom and reproduce with similar quality the thermal fluctuations of proteins but present drastic differences in their agreement with conformational changes. We show that these differences are related to the extent of the deviations from the harmonic approximation, assessed through an anharmonic energy function whose harmonic approximation coincides with the TNM. Our results indicate that mode anharmonicity is more strongly related to its collectivity, i.e., the number of atoms displaced by the mode, than to its amplitude; low-frequency modes can remain harmonic even at large amplitudes, provided they are sufficiently collective. Finally, we assess the potential benefits of different strategies to minimize the impact of anharmonicity. The reduction of the number of degrees of freedom or their regularization by a torsional harmonic potential significantly improves the collectivity and harmonicity of normal modes and the agreement with conformational changes. In contrast, the correction of normal mode frequencies to partially account for anharmonicity does not yield substantial benefits. The TNM program is freely available at https://github.com/ugobas/tnm.     

IFN-γ-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain

It is well established that IFN-γ is required for the development of experimental cerebral malaria (ECM) during Plasmodium berghei ANKA infection of C57BL/6 mice. However, the temporal and tissue-specific cellular sources of IFN-γ during P. berghei ANKA infection have not been investigated, and it is not known whether IFN-γ production by a single cell type in isolation can induce cerebral pathology. In this study, using IFN-γ reporter mice, we show that NK cells dominate the IFN-γ response during the early stages of infection in the brain, but not in the spleen, before being replaced by CD4(+) and CD8(+) T cells. Importantly, we demonstrate that IFN-γ-producing CD4(+) T cells, but not innate or CD8(+) T cells, can promote the development of ECM in normally resistant IFN-γ(-/-) mice infected with P. berghei ANKA. Adoptively transferred wild-type CD4(+) T cells accumulate within the spleen, lung, and brain of IFN-γ(-/-) mice and induce ECM through active IFN-γ secretion, which increases the accumulation of endogenous IFN-γ(-/-) CD8(+) T cells within the brain. Depletion of endogenous IFN-γ(-/-) CD8(+) T cells abrogates the ability of wild-type CD4(+) T cells to promote ECM. Finally, we show that IFN-γ production, specifically by CD4(+) T cells, is sufficient to induce expression of CXCL9 and CXCL10 within the brain, providing a mechanistic basis for the enhanced CD8(+) T cell accumulation. To our knowledge, these observations demonstrate, for the first time, the importance of and pathways by which IFN-γ-producing CD4(+) T cells promote the development of ECM during P. berghei ANKA infection.     

Pancreatic cancer cells and normal pancreatic duct epithelial cells express an autocrine catecholamine loop that is activated by nicotinic acetylcholine receptors α3, α5, and α7

Pancreatic cancer is the fourth leading cause of cancer deaths in developed countries. Smoking is an established risk factor for this malignancy but the underlying mechanisms are poorly understood. Previous reports have provided evidence that nicotinic acetylcholine receptors (nAChR) and beta adrenergic receptors (β-AR) stimulate the growth and migration of pancreatic cancer cells. However, a potential cooperation of these two receptor families in the regulation of pancreatic cancer has not been studied to date. Using two pancreatic cancer cell lines and immortalized pancreatic duct epithelia in vitro, our current data show that all three cell lines synthesized and released the catecholamine neurotransmitters noradrenaline and adrenaline upon exposure to nicotine and that this activity was regulated by α3, α5, and α7-nAChRs. In accordance with the established function of these catecholamines as β-AR agonists, nicotine-induced cell proliferation was blocked by the β-AR antagonist propranolol. Nicotine-induced proliferation was also abolished by the α7-nAChR antagonist α-bungarotoxin, whereas catecholamine production in response to nicotine was blocked by gene knockdown of the α3, α5, and α7-nAChRs. The nicotinic agonists acetylcholine, nicotine, and its nitrosated carcinogenic derivative NNK induced the phosphorylation of CREB, ERK, Src, and AKT and these responses were inhibited by propranolol. Our findings identify this hitherto unknown autocrine catecholamine loop as an important regulatory cascade in pancreatic cancer that may prove a promising new target for cancer intervention.