The circulating blood normally contains no more than 1-2% of the body's population of leucocytes. The numbers and phenotypes of circulating leucocyte subsets can change dramatically during and immediately following exercise. The surface expression of adhesion molecules makes an important contribution to such responses by changing patterns of cell trafficking. Alterations in the surface expression of adhesion molecules could reflect a shedding of molecules, selective apoptosis or differential trafficking of cells with a particular phenotype, effects from mechanical deformation of the cytoplasm, active biochemical processes involving cytokines, catecholamines, glucocorticoids or other hormones, or changes in the induction of adhesion molecules. The expression of adhesion molecules changes with maturation and activation of leucocytes. Typically, mature cells express lower densities of L-selectin (CD62L), the homing receptor for secondary lymphoid organs, and higher densities of LFA-1 (CD11a), the molecule associated with trafficking to non-lymphoid reservoir sites. The neutrophils and natural killer cells that are mobilised during exercise also express high levels of Mac-1 (CD11b), a marker associated with cellular activation. Possibly, exercise demarginates older cells that are awaiting destruction in the spleen. Plasma concentrations of catecholamines rise dramatically with exercise, and there is growing evidence that catecholamines, acting through a cyclic adenosine monophosphate second messenger system, play an important role in modifying the surface expression of adhesion molecules. Analogous changes can be induced by other forms of stress that release catecholamines or by catecholamine infusion, and responses are blocked by beta(2)-blocking agents. Catecholamines also modify adherence and expression of adhesion molecules in vitro. Cell trafficking is modified by genetic deficiencies in the expression of adhesion molecules, but leucocyte responses to exercise and catecholamines are generally unaffected by splenectomy. A number of clinical conditions including atherogenesis and metaplasia are marked by an altered expression of adhesion molecules. The effects of exercise on these molecules could thus have important health implications.

Memory T lymphocytes are commonly viewed as a major barrier for long-term survival of organ allografts and are thought to accelerate rejection responses due to their rapid infiltration into allografts, low threshold for activation, and ability to produce inflammatory mediators. Because memory T cells are usually associated with rejection, preclinical protocols have been developed to target this population in transplant recipients. Here, using a murine model, we found that costimulatory blockade-mediated lung allograft acceptance depended on the rapid infiltration of the graft by central memory CD8+ T cells (CD44(hi)CD62L(hi)CCR7+). Chemokine receptor signaling and alloantigen recognition were required for trafficking of these memory T cells to lung allografts. Intravital 2-photon imaging revealed that CCR7 expression on CD8+ T cells was critical for formation of stable synapses with antigen-presenting cells, resulting in IFN-γ production, which induced NO and downregulated alloimmune responses. Thus, we describe a critical role for CD8+ central memory T cells in lung allograft acceptance and highlight the need for tailored approaches for tolerance induction in the lung.

CD25(High) CD4+ regulatory T cells (Treg cells) have been described as key players in immune regulation, preventing infection-induced immune pathology and limiting collateral tissue damage caused by vigorous anti-parasite immune response. In this review, we summarize data obtained by the investigation of Treg cells in different clinical forms of Chagas' disease. Ex vivo immunophenotyping of whole blood, as well as after stimulation with Trypanosoma cruzi antigens, demonstrated that individuals in the indeterminate (IND) clinical form of the disease have a higher frequency of Treg cells, suggesting that an expansion of those cells could be beneficial, possibly by limiting strong cytotoxic activity and tissue damage. Additional analysis demonstrated an activated status of Treg cells based on low expression of CD62L and high expression of CD40L, CD69, and CD54 by cells from all chagasic patients after T. cruzi antigenic stimulation. Moreover, there was an increase in the frequency of the population of Foxp3+ CD25(High)CD4+ cells that was also IL-10+ in the IND group, whereas in the cardiac (CARD) group, there was an increase in the percentage of Foxp3+ CD25(High) CD4+ cells that expressed CTLA-4. These data suggest that IL-10 produced by Treg cells is effective in controlling disease development in IND patients. However, in CARD patients, the same regulatory mechanism, mediated by IL-10 and CTLA-4 expression is unlikely to be sufficient to control the progression of the disease. These data suggest that Treg cells may play an important role in controlling the immune response in Chagas' disease and the balance between regulatory and effector T cells may be important for the progression and development of the disease. Additional detailed analysis of the mechanisms on how these cells are activated and exert their function will certainly give insights for the rational design of procedure to achieve the appropriate balance between protection and pathology during parasite infections.

Ulcerative colitis (UC) and Crohn's disease (CD) are two forms of chronic inflammatory bowel disease. CD4 T cells play a central role in the pathogenesis of both diseases. Smoking affects both UC and CD but with opposite effects, ameliorating UC and worsening CD. We hypothesized that the severity of gut inflammation could be modulated through T cell nicotinic acetylcholine receptors (nAChRs) and that the exact clinical outcome would depend on the repertoire of nAChRs on CD4 T cells mediating each form of colitis. We measured clinical and immunologic outcomes of treating BALB/c mice with oxazolone- and trinitrobenzene sulfonic acid (TNBS)-induced colitides by nicotine. Nicotine attenuated oxazolone colitis, which was associated with an increased percentage of colonic regulatory T cells and a reduction of Th17 cells. TCR stimulation of naive CD4(+)CD62L(+) T cells in the presence of nicotine upregulated expression of Foxp3. In marked contrast, nicotine worsened TNBS colitis, and this was associated with increased Th17 cells among colonic CD4 T cells. Nicotine upregulated IL-10 and inhibited IL-17 production, which could be abolished by exogenous IL-12 that also abolished the nicotine-dependent upregulation of regulatory T cells. The dichotomous action of nicotine resulted from the up- and downregulation of anti-inflammatory α7 nAChR on colonic CD4 T cells induced by cytokines characteristic of the inflammatory milieu in oxazolone (IL-4) and TNBS (IL-12) colitis, respectively. These findings help explain the dichotomous effect of smoking in patients with UC and CD, and they underscore the potential for nicotinergic drugs in regulating colonic inflammation.

OBJECTIVE

Formation of platelet-leukocyte aggregates (PLAs) is increased in several inflammatory and thrombotic conditions. This may result from and enhance platelet and neutrophil activation and could contribute to the inflammatory process in inflammatory bowel disease (IBD). We investigated platelet-leukocyte aggregation in patients with IBD and its relation to treatment, disease activity and platelet and neutrophil activation.

METHODS

PLAs, platelet activation (P-selectin expression) and neutrophil activation (L-selectin expression) were assessed 30 and 180 minutes after drawing blood into EDTA/citrate-theophylline-adenosine and dipyridamole, a novel anticoagulant, using fluorescent antibodies to CD45 (for leukocytes), CD42a (for platelets), CD62P (P-selectin) and CD62L (L-selectin) and flow cytometry. Platelet activation was also measured using the ADVIA 120 hematology analyser.

RESULTS

Samples from 67 patients with IBD measured within 30 minutes had a higher platelet count (P < 0.001), more platelets expressing P-selectin (P = 0.01), and more PLAs (P < 0.01) than from 20 healthy controls and more PLAs (P < 0.05) than from 9 controls with inflammatory arthropathies. IBD patients on thiopurines had fewer PLAs than those not taking them (P < 0.05); corticosteroids and aminosalicylates had no such effects. Incubation for 180 minutes increased the number of platelets expressing P-selectin (P < 0.0001), and the number of PLAs (P < 0.0001). The PLAs correlated with the number of platelets expressing P-selectin before (r=+0.40, P < 0.001) and after (r=+0.66, P < 0.0001) incubation.

CONCLUSIONS

The number of PLAs is higher in patients with IBD than in healthy and inflammatory controls, but their numbers are lowered by thiopurines. Increased PLA formation may in part be due to increased platelet activation and could be pathogenic in IBD.

Here we show that the genes for perforin, the three major T cell granzymes (A-C) and IFN-gamma are differentially expressed during primary activation of naive CD8(+) T cells, kinetically and at the single-cell level. When CD44(low)CD62L(high)CD8(+) lymph node T cells were activated with IL-2 and immobilized antibodies to CD3, CD8 and CD11a, expression of perforin, granzyme B and IFN-gamma mRNAs was induced by day 2, and increased in parallel with perforin-dependent cytolytic activity. Granzyme C and A transcripts were not detected until 1 and 3 days later respectively. Single-cell PCR showed that expression frequencies rose in parallel with total levels of each mRNA, but that individual cells expressed diverse combinations of perforin, granzyme A-C and IFN-gamma mRNAs. These expression patterns indicated that the delayed expression of granzymes A and C was not due to late activation of distinct cell subpopulations. Statistical analysis of the data suggested that each gene was differentially regulated at the single-cell level. Individual naive CD8(+) T cells gave rise over 7 days to clones that expressed all five products at the clonal level, but also expressed diverse combinations at the single-cell level. We conclude that, during primary activation, CD8(+) T cells progressively acquired the ability to express most or all of these genes, and that the variable expression patterns observed among single cells within clones and populations reflected transient rather than heritable differences in expression profile.

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)

There is increasing interest in the role of blood polymorphonuclear leukocytes (PMNs) in the pathogenesis of sickle cell crisis. We studied the adherence of PMNs from 18 sickle cell patients in crisis, 25 out of crisis, and 43 healthy subjects (controls) to monolayers of human umbilical cord endothelium that were either untreated or pretreated with tumor necrosis factor alpha (TNFalpha). Overall, the PMNs from patients in crisis were more adherent than control PMNs to untreated endothelial monolayers (mean 53% increase; P < .001) and TNFalpha-treated monolayers (mean 41% increase; P < .002). Increased adhesiveness was not associated with an abnormal expression of CD11a, CD11b, CD11c, CD18, CD62L, or CD15. There was an increase in the number of PMNs expressing CD64 in patients in crisis (median value, 44%) compared with patients out of crisis (median, 21%; P = .025) and controls (median, 6.5%; P < .001). Sera from patients in crisis had normal levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-gamma, TNFalpha, interleukin-1 (IL-1), IL-6, or IL-8 and did not modify the adherence of PMNs or their expression of CD64. Only IFN-gamma induced CD64 expression on PMNs, but this effect was not associated with enhanced binding to endothelium. Because PMNs bound to endothelial monolayers were CD64(+) and CD64-enriched PMNs were 7 times more adherent to endothelial monolayers than CD64-depleted PMNs, it is likely that CD64 is a marker of adherent PMNs. Two of the three anti-CD64 antibodies used in our antibody blocking studies (clones 32.2 and 197) partially inhibited the binding of sickle cell PMNs to untreated endothelium (mean inhibitions of 33% [P = .01] and 21% [P = .03], respectively), whereas only one (clone 197) inhibited binding to TNFalpha-treated endothelium (mean inhibition, 29%; P = . 004). In some patients with sickle cell disease, an enhanced PMN adhesion to vascular endothelium could contribute to the vascular occlusion that characterizes the acute crisis of the disease.

Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-gamma production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn's disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn's disease induced by transfer of CD62L+ CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264.7 macrophages. Local administration of the Ad-asIL-18 vector to SCID mice with established colitis led to transduction of epithelial cells and caused a significant suppression of colitis activity, as assessed by a newly developed endoscopic analysis system for colitis. Furthermore, treatment with Ad-asIL-18 induced a significant suppression of histologic colitis activity and caused suppression of mucosal IFN-gamma production, whereas IFN-gamma production by spleen T cells was unaffected. Taken together, these data indicate an important role for IL-18 in the effector phase of a T cell-dependent murine model of colitis and suggest that strategies targeting IL-18 expression may be used for the treatment of patients with Crohn's disease.

L-selectin mediates leukocyte rolling on vascular endothelium at sites of inflammation and lymphocyte migration to peripheral lymph nodes. L-selectin is rapidly shed from the cell surface after leukocyte activation by a proteolytic mechanism that cleaves the receptor in a membrane proximal extracellular region. This process may allow rapid leukocyte detachment from the endothelial surface before entry into tissues. In this study, the structural requirements for regulation of human L-selectin endoproteolytic release were examined through analysis of chimeric selectin molecules and mutant L-selectin receptors. The use of chimeric selectins and a cytoplasmic tail truncation mutant demonstrated that the extracellular membrane-proximal 15-amino acid region of L-selectin is required for endoproteolytic release. The introduction of alanine-scanning mutations within this membrane-proximal region did not prevent endoproteolytic release, indicating that a specific amino acid motif was not an absolute requirement for cleavage. Furthermore, alterations within the putative primary cleavage site (K283-S284) resulted in either constitutive endoproteolytic release of the receptor or inhibition of cell activation-induced shedding to variable extents. The length of the membrane-proximal region was also critical since truncations of this region completely abolished endoproteolytic release. Thus, release of L-selectin is likely to be regulated by the generation of an appropriate tertiary conformation within the membrane-proximal region of the receptor which allows recognition by a membrane-bound endoprotease with relaxed sequence specificity that cleaves the receptor at a specific distance from the plasma membrane. These observations suggest a generalized protein-processing pathway involved in the endoproteolytic release of specific transmembrane proteins which harbor widely differing primary sequences at or neighboring their cleavage sites.

A substantial proportion of patients with acute graft-versus-host disease (aGVHD) respond to cell therapy with culture-expanded human bone marrow mesenchymal stromal/stem cells (BM-MSCs). However, the mechanisms by which these cells can ameliorate aGVHD-associated complications remain to be clarified. We show here that BM-MSC-derived extracellular vesicles (EVs) recapitulated the therapeutic effects of BM-MSCs against aGVHD. Systemic infusion of human BM-MSC-derived EVs prolonged the survival of mice with aGVHD and reduced the pathologic damage in multiple GVHD-targeted organs. In EV-treated GVHD mice, CD4+ and CD8+ T cells were suppressed. Importantly, the ratio of CD62L-CD44+ to CD62L + CD44- T cells was decreased, suggesting that BM-MSC-derived EVs suppressed the functional differentiation of T cells from a naive to an effector phenotype. BM-MSC-derived EVs also preserved CD4 + CD25 + Foxp3+ regulatory T cell populations. In a culture of CD3/CD28-stimulated human peripheral blood mononuclear cells with BM-MSC-derived EVs, CD3+ T cell activation was suppressed. However, these cells were not suppressed in cultures with EVs derived from normal human dermal fibroblasts (NHDFs). NHDF-derived EVs did not ameliorate the clinical or pathological characteristics of aGVHD in mice, suggesting an immunoregulatory function unique to BM-MSC-derived EVs. Microarray analysis of microRNAs in BM-MSC-derived EVs versus NHDF-derived EVs showed upregulation of miR-125a-3p and downregulation of cell proliferative processes, as identified by Gene Ontology enrichment analysis. Collectively, our findings provide the first evidence that amelioration of aGVHD by therapeutic infusion of BM-MSC-derived EVs is associated with the preservation of circulating naive T cells, possibly due to the unique microRNA profiles of BM-MSC-derived EVs. Stem Cells 2018;36:434-445.

The frequency, subsets and activation status of peripheral blood invariant NKT (iNKT) cells were evaluated in pulmonary tuberculosis (TB) patients and in chronically HIV-1-infected subjects. The absolute numbers of iNKT cells were significantly decreased in TB patients and in HIV-1+ individuals who were antiretroviral therapy naive or had detectable viremia despite receiving HAART. iNKT cell subset analysis demonstrated a decreased percentage of CD4(+) iNKT cells in HIV-1+ subjects, and a decreased percentage of double negative iNKT cells in TB patients. Peripheral blood iNKT cells from HIV-1+ and TB patients had significantly increased expression of CD69, CD38, HLA-DR, CD16, CD56, and CD62L. The expression of CD25 was significantly increased only on iNKT cells from TB patients. These findings indicate that peripheral blood iNKT cells in these two chronic infections show an up-regulated expression of activation markers, suggesting their role in the immune response to infection.

Plasmacytoid dendritic cell (PDC)-derived IFN-alpha plays a central role in antiviral defense and in Th1-driven autoimmune diseases, such as systemic lupus erythematosus (SLE). In the current study, we explored how PGE2 effects the phenotype of PDCs from healthy and SLE subjects. Although PGE2 is considered to mediate mainly proinflammatory effects, we show that PGE2 and PG analogs potently inhibit secretion of IFN-alpha by TLR-activated PDCs. This effect is mainly mediated by PG receptors E-prostanoid 2 and E-prostanoid 4 and involves inhibition of IFN regulatory factor 7 expression. Of note, profound IFN-alpha inhibition by PGE2 is also seen in PDCs from SLE subjects, independent of age, disease activity, and therapy. We show that TLR9-activated PDCs treated with PGE2 exhibit DC2-like characteristics with enhanced expression of CD86 and CD62L, and decreased expression of CD80 and MHC class I. Consequently, PGE2-treated PDCs suppress secretion of Th1 cytokines by T cells while increasing the secretion of Th2 cytokines. Prevention of CpG-induced CD62L downregulation by PGE2 suggests that it may induce the retreat of PDCs from inflamed tissues. Our data on the effects of PGE2 on PDCs may explain occasional reports about the induction of SLE-like symptoms by cyclooxygenase inhibitors as well as improvement of such symptoms by treatment with PG analogs. In conclusion, our data suggest that PGE2 and certain PG analogs, some of which are already in clinical use, should be evaluated as a novel and inexpensive treatment approach for patients with SLE and other IFN-alpha-dependent, Th1-driven autoimmune diseases.

TGF-beta induces Foxp3 expression in stimulated T cells. These Foxp3+ cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3+ regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-beta up-regulated Foxp3 in CD4+Foxp3- T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1-2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-beta led to a loss of Foxp3 expression after an approximately 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3- cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3- cells were detected by 28 days after transfer. However, some Foxp3+ cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3+ iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3- cells, Foxp3+ iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in naïve-stimulated T cells is transient in vitro, dependent on TGF-beta activity within a highly restricted window after activation and continuous TGF-beta presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.

BACKGROUND

Acute respiratory distress syndrome (ARDS) affects over 200000 people annually in the USA. Despite causing severe, and often refractory, hypoxaemia, the high mortality and long-term morbidity of ARDS results mainly from extra-pulmonary organ failure; however the mechanism for this organ crosstalk has not been determined.

METHODS

Using autologous radiolabelled neutrophils we investigated the pulmonary transit of primed and unprimed neutrophils in humans. Flow cytometry of whole blood samples was used to assess transpulmonary neutrophil priming gradients in patients with ARDS, sepsis and perioperative controls.

RESULTS

Unprimed neutrophils passed through the lungs with a transit time of 14.2 s, only 2.3 s slower than erythrocytes, and with <5% first-pass retention. Over 97% of neutrophils primed ex vivo with granulocyte macrophage colony-stimulating factor were retained on first pass, with 48% still remaining in the lungs at 40 min. Neutrophils exposed to platelet-activating factor were initially retained but subsequently released such that only 14% remained in the lungs at 40 min. Significant transpulmonary gradients of neutrophil CD62L cell surface expression were observed in ARDS compared with perioperative controls and patients with sepsis.

CONCLUSIONS

We demonstrated minimal delay and retention of unprimed neutrophils transiting the healthy human pulmonary vasculature, but marked retention of primed neutrophils; these latter cells then 'deprime' and are re-released into the systemic circulation. Further, we show that this physiological depriming mechanism may fail in patients with ARDS, resulting in increased numbers of primed neutrophils within the systemic circulation. This identifies a potential mechanism for the remote organ damage observed in patients with ARDS.

Although the pathogenesis of the acute respiratory distress syndrome (ARDS) is complex and poorly understood, several observations point to an important role of interactions between polymorphonuclear neutrophils (PMN) and cytokines in this process. We therefore studied certain parameters involved in PMN transendothelial migration (adhesion molecule expression and cytoskeletal organization) in patients with ARDS (n = 14) in comparison with other ventilated patients (n = 15). We found that in the basal state, both whole-blood PMN and alveolar PMN obtained by bronchoalveolar lavage (BAL) were activated, as shown by decreased L-selectin CD62L and increased beta 2 integrin CD11b expression, as well as decreased F-actin content. The degree of PMN activation increased with the degree of lung injury and with the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8). Moreover, the capacity of ex vivo stimulation of alveolar PMN by a bacterial peptide was low in ARDS and could partly account for the high susceptibility of these patients to lung infection. Therefore, ARDS-associated lung injury could be caused, at least in part, by inappropriate adhesion and transendothelial migration of proinflammatory cytokine-primed PMN.

BACKGROUND

The high-affinity receptor for IgE (FcepsilonRI) on myeloid dendritic cells has been shown to play a major role in atopic dermatitis (AD). Plasmacytoid dendritic cells (pDCs), which are instrumental in the defense of viral infections, are present in reduced amounts in the skin of patients with AD, which is characterized by a high susceptibility to viral infections.

OBJECTIVE

We explored phenotypical and functional characteristics of pDC in the peripheral blood of patients with AD and healthy individuals.

METHODS

Blood dendritic cell antigen-2+CD123+ pDCs were enriched from the peripheral blood of patients with AD and studied in functional assays.

RESULTS

Skin-homing molecules such as cutaneous lymphocyte antigen and L-selectin CD62L were expressed in lower levels on pDCs of patients with AD. pDCs expressed high amounts of IgE-occupied FcepsilonRI. Further, FcepsilonRI aggregation on pDCs impaired the surface expression of MHC I and II, induced the production of IL-10, and enhanced the apoptosis of pDCs. Importantly, FcepsilonRI preactivated pDC produced less IFN-alpha and IFN-beta after stimulation with CpG motifs and enhanced the outcome of immune responses of the TH2 type.

CONCLUSIONS

From these data, we conclude that FcepsilonRI-bearing pDCs from patients with AD (1) are different from pDCs of healthy individuals, (2) might be important in the pathophysiology of AD, and (3) contribute to the enhanced susceptibility of patients with AD to viral infections.

OBJECTIVE

To understand whether directly measured psoriasis severity is associated with vascular inflammation assessed by (18)F-fluorodeoxyglucose positron emission tomography computed tomography.

METHODS

In-depth cardiovascular and metabolic phenotyping was performed in adult psoriasis patients (n=60) and controls (n=20). Psoriasis severity was measured using psoriasis area severity index. Vascular inflammation was measured using average aortic target-to-background ratio using (18)F-fluorodeoxyglucose positron emission tomography computed tomography.

RESULTS

Both the psoriasis patients (28 men and 32 women, mean age 47 years) and controls (13 men and 7 women, mean age 41 years) were young with low cardiovascular risk. Psoriasis area severity index scores (median 5.4; interquartile range 2.8-8.3) were consistent with mild-to-moderate skin disease severity. Increasing psoriasis area severity index score was associated with an increase in aortic target-to-background ratio (β=0.41, P=0.001), an association that changed little after adjustment for age, sex, and Framingham risk score. We observed evidence of increased neutrophil frequency (mean psoriasis, 3.7±1.2 versus 2.9±1.2; P=0.02) and activation by lower neutrophil surface CD16 and CD62L in blood. Serum levels of S100A8/A9 (745.1±53.3 versus 195.4±157.8 ng/mL; P<0.01) and neutrophil elastase-1 (43.0±2.4 versus 30.8±6.7 ng/mL; P<0.001) were elevated in psoriasis. Finally, S100A8/A9 protein was related to both psoriasis skin disease severity (β=0.53; P=0.02) and vascular inflammation (β=0.48; P=0.02).

CONCLUSIONS

Psoriasis severity is associated with vascular inflammation beyond cardiovascular risk factors. Psoriasis increased neutrophil activation and neutrophil markers, and S100A8/A9 was related to both skin disease severity and vascular inflammation.

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9-14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/-) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(-) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/-)CD69(+)NCR(high)CD62L(-) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.

CD62L/L-selectin is a marker found on naïve T cells and further distinguishes central memory (Tcm, CD62L+) from effector memory (Tem, CD62L-) T cells. The regulation of CD62L plays a pivotal role in controlling the traffic of T lymphocytes to and from peripheral lymph nodes. CD62L is shed from the cell membrane following T cell activation, however, the physiological significance of this event remains to be elucidated. In this study, we utilized in vitro generated anti-tumor antigen T cells and melanoma lines as a model to evaluate the dynamics of CD62L shedding and expression of CD107a as a marker of lytic activity. Upon encounter, with matched tumor lines, antigen reactive T cells rapidly lose CD62L expression and this was associated with the acquisition of CD107a. By CD62L ELISA, we confirmed that this transition was mediated by the shedding of CD62L when T cells encountered specific tumor antigen. The introduction of a shedding resistant mutant of CD62L into the tumor antigen-reactive T cell line JKF6 impaired CD107a acquisition following antigen recognition and this was correlated with decreased lytic activity as measured by (51)Cr release assays. The linkage of the shedding of CD62L from the surface of anti-tumor T cells and acquisition of lytic activity, suggests a new function for CD62L in T cell effector functions and anti-tumor activity.

T-cell receptor (TCR) gene therapy enables for the rapid creation of antigen-specific T cells from mice of any strain and represents a valuable tool for preclinical immunotherapy studies. Here, we describe the superiority of γ-retroviral vectors compared with lentiviral vectors for transduction of murine T cells and surprisingly illustrate robust gene-transfer into phenotypically naive/memory-stem cell like (TN/TSCM; CD62L(hi)/CD44(low)) and central memory (TCM; CD62L(hi)/CD44(hi)) CD8+ T cells using murine stem cell-based γ-retroviral vectors (MSGV1). We created MSGV1 vectors for a major histocompatibility complex-class I-restricted TCR specific for the melanocyte-differentiation antigen, glycoprotein 100 (MSGV1-pmel-1), and a major histocompatibility complex-class II-restricted TCR specific for tyrosinase-related protein-1 (MSGV1-TRP-1), and found that robust gene expression required codon optimization of TCR sequences for the pmel-1 TCR. To test for functionality, we adoptively transferred TCR-engineered T cells into mice bearing B16 melanomas and observed delayed growth of established tumors with pmel-1 TCR engineered CD8+ T cells and significant tumor regression with TRP-1 TCR transduced CD4 T cells. We simultaneously created lentiviral vectors encoding the pmel-1 TCR, but found that these vectors mediated low TCR expression in murine T cells, but robust gene expression in other murine and human cell lines. These results indicate that preclinical murine models of adoptive immunotherapies are more practical using γ-retroviral rather than lentiviral vectors.

The transcription factor Kruppel-like factor 2 (KLF2) was proposed to regulate genes involved in cell cycle entry and T cell trafficking; however, the physiological role of its expression in postactivated T cells is not well defined. Previous studies suggested that the cytokines IL-2 and IL-15 differentially regulate KLF2 re-expression in postactivation T cells and that these cytokines also influence effector versus memory T cell differentiation. Using conditional and inducible KLF2-knockout model systems, we tested the specific role of KLF2 expression in activated CD8(+) T cells cultured with these cytokines. KLF2 was required for effective transcription of sphingosine-1-phosphate receptor-1 (S1P(1)) and CD62L in postactivation T cells. However, although different cytokines dramatically altered the expression of cell-cycle-related genes, endogenous KLF2 had a minimal impact. Correspondingly, KLF2-deficient T cells showed dysregulated trafficking but not altered proliferative characteristics following in vivo responses to Ag. Thus, our data help to define KLF2-dependent and -independent aspects of activated CD8(+) T cell differentiation and argue against a physiological role in cell cycle regulation.

Healthy liver, intestine, lung, and skin harbor resident lymphocytes with conventional and unconventional phenotypes. Lymphocytes also have been detected in healthy mice kidneys; however, these cells have not been well studied and have been largely overlooked. To better characterize the intra-renal lymphocytes, we extensively perfused C57BL/6J mice with PBS and then isolated mononuclear cells for flow cytometry analysis. We observed T cells, B cells, and NK cells in normal mice kidneys after extensive perfusion. Approximately 50% of kidney T lymphocytes expressed intermediate levels of CD3 (CD3int T cells). Similar to liver and lung, a high percentage of unconventional CD3+CD4(-)CD8(-) double-negative T cells was observed in normal mice kidneys, from which 11% expressed B220 antigen. Unlike the spleen and blood, the classic CD4+ and CD8+ T lymphocytes in the kidney had a high proportion of activated CD69+ and effector/memory CD44- CD62L ligand phenotypes. Also, a small percentage of CD4+CD25+forkhead box p3+ and NKT cells was observed in perfused and exanguinated kidneys. In addition, a distinct TCR repertoire was found on intra-renal conventional and unconventional T cells compared with those from the spleen. Finally, after 24 h of renal ischemia reperfusion injury (IRI), increased production of cytokines IFN-gamma and TNF-alpha by CD4+ and CD8+ T cells, isolated from perfused kidneys, was observed. These data suggest that some of these cells harbored in the kidney could be implicated in the immune response of the IRI pathogenic process.

BACKGROUND

Recent evidence suggests that morbid obesity is a chronic inflammatory condition that may be associated with immune dysfunction. To test this hypothesis, we investigated several leukocyte cell surface markers of chronic inflammation and followed their response to surgically-induced weight loss.

METHODS

26 patients having Roux-en-Y gastric bypass (RYGBP) for morbid obesity (BMI>> 40) were compared to 10 normal controls (BMI < 25). Relative monocyte and neutrophil frequencies and expression of the activation antigens CD11b (adhesion molecule), CD16 (Fc receptor), and CD62L (L-selectin), were evaluated by flow cytometry preoperatively and at 1, 3, 6 and 12 months after RYGBP. Cases served as their own controls but were also compared to non-obese controls. The results were statistically analyzed using Student's t-test and ANOVA for parametric values and Mann-Whitney along with Kruskal-Wallis ANOVA for nonparametric values.

RESULTS

The control group had mean age 37 +/- 7.6 with mean 23 +/- 2.5 and no comorbidities. The mean age of the sample group was 40.36 +/- 13.7 with mean BMI 52 +/- 8.2. The neutrophil and monocyte relative frequencies of CD11b (monocytes and neutrophils), and CD16 (neutrophils only) were comparable to controls at baseline and did not change significantly with weight loss throughout the study period. However, a significant reduction of CD62L (L-selectin) expression was noted in monocytes and neutrophils at baseline (neutrophils 103 vs 240 gmf, p < 0.001) (monocytes 104 vs 246 gmf, P < 0.001) when compared to normal controls. Levels of L-selectin normalized by 6 months in both monocytes and neutrophils, and by 12 months had become abnormally elevated in monocytes (monocytes 391 gmf, P = 0.007); in neutrophils, there was an upward trend that did not reach significance. The expression of the LPS receptor CD14 in the study group was elevated significantly compared to controls at baseline (1129 vs 719 gmf, P = 0.004); this marker appeared to return to normal by 3 months. Monocyte CD14+/CD16+ subset percentage were also elevated significantly at baseline (14.3% vs 5.25%, P < 0.001), declined throughout the time period but was still significant at 1 year (8.8%, P < 0.001). Eosinophil percentages were elevated at baseline (3.3% obese vs 1.8% controls, P = 0.003) and remained so throughout the time period.

CONCLUSIONS

Deficiencies in the immune system of morbidly obese individuals include elevated levels of eosinophils, monocyte CD14, and monocyte CD14+/CD16+ subsets, with depression of monocyte and neutrophil CD62L. These abnormal levels reverse rapidly with surgically-induced weight loss. RYGBP is not only a weight loss operation but also appears to be an immune restorative procedure.