T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.

DNA vaccines or peptides are capable of inducing specific immunity; however, their translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein, we demonstrate that a novel immunization strategy, encompassing sequential exposure of the lymph node milieu to plasmid and peptide in a heterologous prime-boost fashion, results in considerable MHC class I-restricted immunity in mice. Plasmid-primed antigen expression was essential for the generation of a population of central memory T cells, expressing CD62L and low in PD-1, with substantial capability to expand and differentiate to peripheral memory and effector cells, following subsequent exposure to peptide. These vaccine-induced T cells dominated the T cell repertoire, were able to produce large amounts of chemokines and pro-inflammatory cytokines, and recognized tumor cells effectively. In addition to outlining a feasible and effective method to transform plasmid DNA vaccination into a potentially viable immunotherapeutic approach for cancer, this study sheds light on the mechanism of heterologous prime-boost and the considerable heterogeneity of MHC class I-restricted T cell responses.

In most species, γδ T cells preferentially reside in epithelial tissues like the skin. Lymph duct cannulation experiments in cattle revealed that bovine dermal γδ T cells are able to migrate from the skin to the draining lymph nodes via the afferent lymph. For αβ T cells, it is generally accepted that epithelial and mucosal tissue egress is regulated by expression of the CCR7 chemokine receptor. In this study, we tracked the migratory route of bovine lymph-derived γδ T cells and examined their CCR7 cell surface expression in several compartments along this route. Total lymph cells from afferent and efferent origin were labeled with PKH fluorescent dyes and injected into the bloodstream. PKH(+) cells already reappeared in the afferent lymph after 4 h. The vast majority of the PKH(+) cells retrieved from the afferent lymph were of the WC1(+) γδ T cell phenotype, proving that this PKH(+) γδ T cell subset is able to home to and subsequently exit the skin. PKH(+) γδ T cells from afferent and efferent lymph lack CCR7 surface expression and display high levels of CD62L compared with CD4 T cells, which do express CCR7. Skin homing receptors CCR4 and CCR10 in contrast were transcribed by both CD4 and γδ T cells. Our findings suggest that γδ T cell skin egress and migration into the peripheral lymphatics is CCR7-independent and possibly mediated by CD62L expression.

In a model of peritoneal metastasis in immune-competent mice, we show that nuclear factor (NF)-κB inhibition in CT26 colon cancer cells prevents metastasis. NF-κB inhibition, by stable overexpression of IκB-α super-repressor, induced differential polarization of co-cultured macrophages to an M1-like anti-tumour phenotype in vitro. NF-κB-deficient cancer cell-conditioned media (CT26/IκB-α SR) induced interleukin (IL)-12 and nitric oxide (NO) synthase (inducible NO synthase (iNOS)) expression in macrophages. Control cell (CT26/EV) conditioned media induced high levels of IL-10 and arginase in macrophages. In vivo, this effect translated to reduction in metastasis in mice injected with CT26/ IκB-α SR cells and was positively associated with increased CD8(+)CD44(+)CD62L(-) and CD4(+)CD44(+)CD62L(-) effector T cells. Furthermore, inhibition of NF-κB activity induced high levels of NO in infiltrating immune cells and decreases in matrix metalloproteinase-9 expression, simultaneous with increases in tissue inhibitor of metalloproteinases 1 and 2 within tumours. CT26/IκB-α SR tumours displayed increased pro-inflammatory gene expression, low levels of angiogenesis and extensive intratumoral apoptosis, consistent with the presence of an anti-tumour macrophage phenotype. Macrophage depletion reduced tumour size in CT26/EV-injected animals and increased tumour size in CT26/IκB-α SR cells compared with untreated tumours. Our data demonstrate, for the first time, that an important implication of targeting tumour cell NF-κB is skewing of macrophage polarization to an anti-tumour phenotype. This knowledge offers novel therapeutic opportunities for anticancer treatment.

BACKGROUND

Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction.

RESULTS

Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4(+)CD62L(+) cells into RAG1(-/-)-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS-induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1beta in Caco-2-IEC and human intestinal fibroblasts.

CONCLUSIONS

Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1beta is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.

BACKGROUND

The adult human thymus contributes to de novo T cell synthesis; such synthesis can be assessed by analyzing T cell receptor excision circles (TREC).

METHODS

TREC levels were measured in total peripheral blood mononuclear cells (PBMC) and CD4- and CD8-enriched cells of 29 HIV-positive patients with maximal viral suppression. The expression of CD45RA+CD45RO-, CD45RA+CD62L+, CD45RO-CD27+CD95low and HLA-DR+CD38+ was assessed using three-color flow cytometric analysis of whole blood. Thymic index score was based on computed tomographic scans of the thymus. The relationship of TREC with thymic index and the expression of the naive phenotypes was evaluated.

RESULTS

TREC expression was not statistically different in these HIV-positive patients from that in age-matched HIV-negative controls. Among HIV-positive patients with CD4 cell count of>> 500 x 10(6) cells/l after antiretroviral therapy (n = 15), PBMC TREC levels correlated with the expression of CD45RA+CD45RO- and CD45RA+CD62L+ naive phenotypes, and inversely correlated with the expression of HLA-DR+CD38+. The change between pre- and post-therapy CD4 cell counts for these 15 patients significantly correlated with both thymic index and expression of the CD45RA+CD45RO- phenotype.

CONCLUSIONS

The finding that TREC expression was equivalent between HIV-positive patients after therapy and HIV-negative donors suggests that there is no reduction in thymic output among HIV-positive individuals after therapy. Given that TREC is inversely correlated with HLA-DR/CD38 expression, its analysis in studies of thymopoiesis should be evaluated in the context of maximum viral suppression to reduce HIV-mediated immune activation and/or by normalizing for cell turnover.

BACKGROUND

CD8(+) T cell responses develop rapidly during infection and are swiftly reduced during contraction, wherein >90% of primed CD8(+) T cells are eliminated. The role of apoptotic mechanisms in controlling this rapid proliferation and contraction of CD8(+) T cells remains unclear. Surprisingly, evidence has shown non-apoptotic activation of caspase-3 to occur during in vitro T-cell proliferation, but the relevance of these mechanisms to in vivo CD8(+) T cell responses has yet to be examined.

RESULTS

We have evaluated the activity of caspase-3, a key downstream inducer of apoptosis, throughout the entirety of a CD8(+) T cell response. We utilized two infection models that differ in the intensity, onset and duration of antigen-presentation and inflammation. Expression of cleaved caspase-3 in antigen specific CD8(+) T cells was coupled to the timing and strength of antigen presentation in lymphoid organs. We also observed coordinated activation of additional canonical apoptotic markers, including phosphatidylserine exposure. Limiting dilution analysis directly showed that in the presence of IL7, very little cell death occurred in both caspase-3(hi) and caspase-3(low) CD8(+) T cells. The expression of active caspase-3 peaked before effector phenotype (CD62L(low)) CD8(+) T cells emerged, and was undetectable in effector-phenotype cells. In addition, OVA-specific CD8(+) cells remained active caspase-3(low) throughout the contraction phase.

CONCLUSIONS

Our results specifically implicate antigen and not inflammation in driving activation of apoptotic mechanisms without cell death in proliferating CD8(+) T cells. Furthermore, the contraction of CD8(+) T cell response following expansion is likely not mediated by the key downstream apoptosis inducer, caspase-3.

The ability to expand tumour-infiltrating lymphocytes in vitro has been greatly enhanced by the use of antigen-independent mechanisms of immune cell costimulation. We have produced human, using the K562 cell line, and murine, using YAC-1 cells, artificial antigen presenting cells (aAPC) and demonstrate that these cell types stimulate murine lymphocyte populations in distinct ways. Using aAPC that have been transfected with CD137L (4-1BBL) and CD32 (FcRgammaII), as a means to bind anti-CD3 and anti-CD28 antibody, we found that CD4 cells preferentially expanded in vitro with K562 aAPC, while CD8 cells expanded with both K562 and YAC-1 aAPC. Co-stimulation mediated by CD137L on aAPC was superior to that mediated by anti-CD28 antibody. This was seen in both long and short-term expansion assays, and by the rapid induction of a CD8+ DX5+ population. DX5 serves, under these in vitro conditions, as a general marker for lymphocyte activation. In vivo, the superiority of CD137L was demonstrated by the induction of T helper 1 effectors seen in freshly isolated splenocytes from mice immunized with CD137L-expressing neuroblastoma tumour vaccines. The ability to stimulate a strong CD8 CTL response in vivo correlated with the induction of a DX5+ cell population in splenocytes with a memory-effector phenotype. The presence of this unique DX5+ cell population, phenotypically distinct with regards to CD69 and CD62L expression from DX5+ cells induced by aAPC in vitro, may be associated with the ability of CD137L to induce strong anti-tumour immunity.

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.

Immature dendritic cells (DC), in contrast to their mature counterparts, are incapable of mobilizing a CD8+ CTL response, and, instead, have been reported to induce CTL tolerance. We directly addressed the impact of immature vs mature DC on CTL responses by infusing adenovirus peptide-loaded DC (of the D1 cell line) into mice that had received adenovirus-specific naive TCR-transgenic CD8+ T cells. Whereas i.v. injection of mature DC triggered vigorous CTL expansion, immature DC elicited little proliferation involving only a minority of the TCR-transgenic CTL. Even though the latter CTL developed effector functions, including cytolytic activity and proinflammatory cytokine secretion, these cells differed significantly from CTL primed by mature DC in that they did not exhibit down-regulation of CD62L and CCR7, receptors involved in trapping of T cells in the lymphoid organs. Interestingly, adoptive transfer of CTL effector cells harvested after priming by either mature or immature DC into naive recipient mice, followed by exposure to adenovirus, yielded quantitatively and qualitatively indistinguishable CTL memory responses. Therefore, in vivo priming of naive CD8+ T cells by immature DC, although failing to induce a full-blown, systemic CTL response, resulted in the formation of central memory-like T cells that were able to expand and produce IFN-gamma upon secondary antigenic stimulation.

CD4+CD25(high)CD127(low/neg) regulatory T cells (Tregs) play a critical role in the maintenance of peripheral tolerance and in controlling the development of autoimmune diseases. A combination of surface and intracellular markers, namely, CD25, CD39/CD73, CD62L, CD45RO, CD127, glucocorticoid-induced tumor necrosis factor receptor (GITR), CTLA-4, and the forkhead/winged helix transcription factor (FOXP3), has been used to characterize Tregs. Tregs suppress T effector responses mainly in a direct cell-cell contact manner. However, other mechanisms independent from this manner cannot be excluded entirely. It has been shown that Tregs can undergo limited expansion in vitro after the stimulation of TCR in the presence of exogenous cytokines, e.g., IL-2. Expanded Tregs retain their suppression function. Human Tregs have demonstrated their great potential to be used as a therapeutic intervention in preventing graft rejection and treating autoimmune diseases. In this chapter, we have given a review on how to characterize, isolate, expand Tregs and assess their suppressive functions.

The transforming growth factor (TGF)-β/β-catenin pathway is involved in granulocytic myeloid-derived suppressor cell (G-MDSCs)-induced Foxp3 expression in CD4(+)CD25(-)T cells, which plays an essential role in maintaining feto-maternal tolerance.

Decidual G-MDSCs play an important role in promoting Foxp3 induction in CD4(+)CD25(-)T cells, which is dependent on TGF-β/β-catenin pathway.

MDSCs contribute to the observed increase in regulatory T cells in animal cancer models. The TGF-β/β-catenin pathway is required for T cell development and survival.

MDSC levels in deciduas from patients undergoing elective termination of pregnancy or spontaneous abortion were assessed by flow-cytometric analysis. The best characterized markers of G-MDSCs cells were examined by immunocytochemistry and flow-cytometric analysis. In vivo, fetus resorption and proportion of decidual immune cells were evaluated after depletion of G-MDSCs. In vitro, we established an antigen-non-specific (CD3/CD28) CD4(+)CD25(-)T and G-MDSC co-culture system and added TGF-β, anti-TGFβ, TGF-β plus anti-TGFβ or β-catenin inhibitor ICG001 to the system. Protein levels were measured by western blot.

G-MDSCs showed a significant decrease in spontaneous abortion compared with elective abortion in women with normal pregnancy (P < 0.01), whereas the numbers of monocytic MDSCs remained unchanged. The dynamics of G-MDSCs in mice revealed that few G-MDSCs were present in non-pregnant uteri. G-MDSCs expanded rapidly in CBA/J×BALB/c mice with normal pregnancy and decreased in CBA/J×DBA/2 mice with abortion-prone pregnancy. G-MDSCs were characterized by the expression of CD115, CD117, CD135, CD62L, CCR2, MHCII, CD80, Arginase I and iNOS, and a lack of F4/80 or CD11c expression. Specifically, depletion of G-MDSCs-induced severe embryo resorption and decreased the percentage of CD4(+)CD25(+)Foxp3(+)T cells. In vitro, G-MDSCs had an important role in promoting Foxp3 induction in CD4(+)CD25(-)T cells, dependent on TGF-β/β-catenin pathway.

It is not sufficient to examine the role of G-MDSCs in the maintenance of maternal-fetal tolerance by depleting G-MDSCs using neutralizing antibody. Further studies are needed to establish an animal model of G-MDSCs in order to elucidate their exact role at the maternal-fetal tolerance.

Our findings provide novel insights into a new function and mechanism of action for G-MDSCs in mediating feto-maternal immune tolerance.

Not applicable.

This research was supported by the National Natural Science Foundation of China (Grant No. 81270715; 91442113). The authors have nothing to disclose.

CD44 and l-selectin (CD62L) are major adhesion receptors that mediate leucocyte recruitment at inflammatory sites and lymph nodes, by supporting cell rolling under blood flow. Both CD44 and CD62L have been implicated in inflammatory skin disorders, but their specific involvement in an immediate-type allergic reaction remains uncertain. We used mice deficient in CD44 or CED62L or both in order to determine whether one or both of these molecules were required for leucocyte extravasation in an atopic dermatitis-like allergic response. Wild-type (WT) mice and mice deficient in CD44, CD62L or both were immunized with ovalbumin (OVA). Inflammatory reaction in the ear was elicited once by means of intradermal injection of OVA. Effective sensitization of CD62L knockout (KO) mice required intraperitoneal antigen injection; however, OVA-specific T helper 2 (Th2)-type immune responses and IgE production in mice lacking CD44, CD62L or both were comparable to those in WT mice following intraperitoneal immunization. We employed intravital videomicroscopy to monitor the recruitment of fluorescence-labelled leucocytes to the ear tissue following challenge with OVA. The number of adherent leucocytes was significantly reduced in CD44 KO and CD44/CD62L double KO mice, indicating that CD44 was involved in firm adhesion, the committed step of leucocyte extravasation. Histology of the OVA-challenged ears showed a diminished leucocyte infiltration in the ears of CD44 KO and double KO mice. The results of our study demonstrate that CD44, but not CD62L, is required for leucocyte extravasation during a Th2-type inflammatory response.

OBJECTIVE

The role of T lymphocytes in the pathogenesis of Celiac disease (CD) is well established. However, the mechanisms of T-cell involvement remain elusive. Little is known on the distribution of T subpopulations: T-regulatory (Treg), Th17, CD103, and CD62L cells at disease onset and after gluten-free diet (GFD). We investigated the involvement of several T subpopulations in the pathogenesis of CD.

METHODS

We studied T cells both in the peripheral blood (PB) and the tissue-infiltrating lymphocytes (TILs) from the mucosa of 14 CD patients at presentation and after a GFD, vs. 12 controls.

RESULTS

Our results extend the involvement of Treg, Th1, and Th17 cells in active CD inflammation both in the PB and at the TILs. At baseline, Tregs, Th1, and Th17 cells are significantly higher in active CD patients in TILs and PB. They decreased after diet. Moreover, CD62L+ TILs were increased at diagnosis as compared with GFD patients.

CONCLUSIONS

Our data show significant modifications of the above-mentioned subpopulations both in the PB and TILs. The increase of suppressive Tregs in active CD both in the PB and TILs is intriguing. T lymphocytes are known to have a crucial role in the pathogenesis of CD. We have shown that gluten trigger results in systemic recruitment of T lymphocytes, the unbalance between pro-inflammatory and anti-inflammatory populations and the increase of CD62L+ T cells in TILs. Our results delineate a more complete picture of T-cell subsets in active vs. GFD disease. Our data of T-cell subpopulations, combined with known data on cytokine production, support the concept that duodenal micro-environment acts as an immunological niche and this recognition may have an important role in the diagnosis, prognosis and therapeutical approach of CD.

The impairment of humoral immune response in elderly humans has been extensively demonstrated. We have reported the increase of memory B cells (IgG(+)IgD(-)CD27(-), double negative, DN) population in the elderly, in which there is also a typical inflammatory micro-environment. In order to evaluate whether this pro-inflammatory status could influence the trafficking phenotype of naïve/memory B cells, we have assessed the expression of CCR7, CCR6, CXCR3, CXCR4, CXCR5 and CD62L on naïve/memory B cell subpopulations in young and elderly subjects. Moreover, the combination of pro-inflammatory interleukin-21 (IL-21) and B cell receptor (BCR) stimulation enables B cells to produce and secrete granzyme B (GrB), which plays a critical role in early anti-viral immune responses, in the regulation of autoimmune mechanisms and in cancer immunosurveillance. Our data demonstrate that in the elderly, naïve/memory B cell populations present a different expression of the studied receptors that could be discussed in terms of "inflamm-aging". In particular IgG(+)IgD(-)CD27(-) DN B cells show a tissue trafficking phenotype and they can be stimulated to produce GrB.

BACKGROUND

Preterm infants with respiratory distress syndrome (RDS) present with systemic inflammation. The role of lymphocytes in RDS is less studied. Activation of lymphocytes could mediate chronic inflammation and development of bronchopulmonary dysplasia (BPD).

OBJECTIVE

To evaluate whether T cells are activated in preterm infants with RDS and whether T cell activation is associated with the development of BPD.

METHODS

Thirty-four infants with RDS [mean gestational age 27.1 (SD 2.0) weeks, birth weight 900 (216) g] were compared with 21 infants without RDS [32.6 (1.4) weeks, 1,697 (406) g]. From blood samples taken on postnatal days 1, 3, and 7, CD4 and CD8 cell counts and their expressions of co-stimulatory molecule CD54 and adhesion molecule CD62L were determined by flow cytometry. In activated cells, expression of CD54 is increased and CD62L is decreased.

RESULTS

As compared with infants without RDS, infants with RDS had less CD4 and CD8 cells on day 3 (both p = 0.02). On day 1 and day 3, RDS was associated with increased CD54 expression on CD4 cells (p = 0.001; p = 0.03) and decreased CD62L expression on CD8 cells (both p = 0.02). Infants with RDS who developed BPD (n = 18) had higher CD54 expression on CD4 cells on day 3 (p = 0.01) and on CD8 cells on day 1 and day 3 (p = 0.01; p = 0.04) as compared with infants without BPD (n = 16).

CONCLUSIONS

In preterm infants, RDS is associated with a lower T cell count and a higher proportion of activated cells. Increased proportion of activated T cells predicts the development of BPD. Systemic T cell activation could mediate inflammation and development of BPD.

BACKGROUND

Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.

METHODS

The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10(-)D16(low) in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.

RESULTS

SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10(-)CD16(low) phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.

CONCLUSIONS

Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56(+) cells induces increased expansion of CD56(+)CD3(-) cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16(low/neg) NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.

The mechanisms involved in impaired immunity in malnourished children are not well understood. CD4(+) CD62L(-) and CD8(+) CD28(-) do not express the naive cell markers CD62L and CD28, suggesting that they function as effector T cells. Using a flow cytometry-based analysis we examined the proportions of CD4(+) CD62L(-) and CD8(+) CD28(-) T cell subsets in well-nourished infected (WNI) and malnourished infected (MNI) children. Here we report that WNI children had a higher percentage of CD4(+) CD62L(-) (11.1 +/- 1.0) and CD8(+) D28(-) (40.2 +/- 5.0) T cell subsets than healthy (6.5 +/- 1.0 and 23.9 +/- 4.8) and MNI children (7.4 +/- 1.1 and 23.1 +/- 6.2, respectively) (P < 0.5). Data suggest that WNI children respond efficiently against pathogenic microbes. In contrast, relatively low numbers of circulating of CD4(+) CD62L(-) and CD8(+) CD28(-) T cells in MNI children may represent an ineffective response to infection. Levels of effector T cells in children with gastrointestinal infections versus those suffering from respiratory infections were also significantly different within the WNI group. While WNI children with gastrointestinal infections had higher absolute and relative values of CD8(+), and CD8(+) CD28(-) T subsets, by those with respiratory infections had higher values of CD4(+) lymphocytes. However, due to the small number of subjects examined, our results in WNI children should be interpreted with caution and confirmed using a larger sample size. Our data suggest that altered expression of CD62L and CD28 receptors may contribute to impaired T cell function observed in MNI children.

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8(+) T(N) cells (CD4(-)CD62L(+)CD45RA(+)) and CD8(+) T(CM) (CD4(-)CD62L(+)CD45RA(-)) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T(N) and T(CM) we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

Neutrophils provide first-line defense against infections and are potent effectors in innate and adaptive immunity. Recently neutrophils have been shown to play important roles in multiple antitumor reactions. A subset of mature neutrophils in human systemic inflammation has been identified as a unique circulating population of myeloid cells, which is capable of inhibiting T cell responses. These neutrophils show unique immunophenotype (CD11c bright/CD62L dim/CD11b bright/CD16 bright). This study reports detection of mature neutrophils with similar immunophenotype in the peripheral blood samples of cancer patients using flow cytometry analysis. This population of neutrophils is not detected in peripheral blood samples of normal controls. Thus this finding suggests the involvement of mature neutrophils in antitumor immunity.

To define the role of memory T cells in a non-persistent viral infection, we have delineated the phenotype of memory CD8+ T cells specific for influenza A virus (FluA; matrix protein M158-66) based on the expression of several memory/effector lineage markers and relevant chemokine receptors. We found a majority of FluA-specific CD8+ T cells expressed CD27 and CD28, and variably expressed CD45RA, CD62L, CD94 and granzyme A. A majority of FluA-specific CD8+ T cells expressed high levels of CXCR3, and moderate levels of CCR5 and CXCR4, whereas a limited proportion expressed CCR7, CCR6 and CXCR5. A phenotypic profile based on these observations showed that there are both immature and mature memory CD8+ T cells specific for FluA.

Here we provide a description of lymphocyte populations in human lymph nodes (LN) with a special emphasis on the CD4+ lymphocyte population constitutively expressing CD25 at a high level and endowed with immunoregulatory properties [T regulatory (Treg) cells]. Lymph nodes were analysed by multicolour flow cytometry in parallel with correspondent peripheral blood (PB). Immunomagnetically purified Treg cells were tested for anergy and suppressive activity in a CD3/T-cell receptor (TCR)-driven proliferation assay. Compared to PB, there was a reduced T/B lymphocyte ratio in LN. Both LN and PB contained a similar proportion of CD4+ lymphocytes but, conversely, CD8+ lymphocytes were less represented in PB, with a consequent increase in the ratio of CD4+/CD8+ natural killer cells were <2% (PB range 6-22%). No significant differences existed in the frequency of the other lymphocyte subpopulations examined (naïve-type CD4+ and CD8+ lymphocytes, activated B and CD4+ lymphocytes, and effector-type CD8+ lymphocytes). LN and PB contained similar percentages of CD4+ lymphocytes constitutively expressing intermediate or high levels of CD25. CD4+ CD25++ cells constitutively coexpressed high levels of CD152 and were therefore identified as Treg cells. Treg cells in LN and PB differed in terms of CD45RB, HLA-DR, CD45RO, and CD62L expression. Also the TCRVbeta repertoire diverged between Treg cells from LN and PB. Similar to Treg cells from PB, Treg cells from LN were anergic and efficiently inhibited other CD4+ and CD8+ lymphocyte proliferation. This study extends the information on the diversities in lymphocyte composition between human LN and PB, and reports for the first time a description of the phenotypic and functional characteristics of Treg cells in human LN, highlighting the importance of the LN microenvironment in shaping the surface phenotype of Treg cells.