gammadelta T lymphocytes recognize non-peptidic microbial antigens without antigen processing and major histocompatibility complex (MHC) restriction, representing an early defence mechanism against invading pathogens. As a defective response to non-peptidic antigens was observed in human immunodeficiency virus-positive (HIV+) persons, the aims of this study were twofold: to analyse the incidence of gammadelta T-cell anergy in HIV+ positive patients with opportunistic infections/co-infections (HIV-OIC), and to investigate the role of highly active antiretroviral therapy (HAART) on gammadelta T-cell functions. Peripheral gammadelta T-cell distribution and in vitro reactivity to a non-peptidic mycobacterial antigen, isopentenyl pyrophosphate (IPP), were analysed. gammadelta T-cell subset distribution was altered more in HIV-OIC patients than in asymptomatic HIV+ subjects (HIV-ASY). Specifically, the Vdelta2/Vdelta1 ratio was inverted as a consequence of a decrease in Vdelta2 T-cell number. Moreover, IPP-stimulated Vdelta2 T cells from the HIV-OIC group displayed a major defect in interferon-gamma (IFN-gamma) production. Interestingly, HAART induced a sustained recovery of naive CD45RA+ and CD62L+ T cells and restored gammadelta T-cell function. Accordingly, in vitro CD45RA depletion resulted in gammadelta T-cell hyporesponsiveness. Altogether, the incidence of gammadelta T-cell anergy was increased in HIV-OIC patients and dependent on CD45RA helper function. Moreover, HAART was able to restore gammadelta T-cell reactivity, extending the immune recovery to non-peptide microbial antigens.

It is generally thought that during the contraction phase of an acute anti-viral T cell reponse, the effector T cells that escape activation-induced cell death eventually differentiate into central memory T cells over the next several weeks. Here we report that antigen-specific CD8T cells with the phenotype and function of central memory cells develop concomitantly with effector T cells during vaccinia virus (vv) infection. As soon as 5 days after an intraperitoneal infection with vv, we could identify a subset of CD44(hi) and CD62L(+) vv-specific CD8 T cells in the peritoneal exudate lymphocytes. This population constituted approximately 10% of all antigen-specific T cells and like central memory T cells, they also expressed high levels of CCR7 and IL-7R but expressed little granzyme B. Importantly, upon adoptive transfer into naïve congenic hosts, CD62L(+), but not CD62L(-) CD8 T cells were able to expand and mediate a rapid recall response to a new vv challenge initiated 6 weeks after transfer, confirming that the CD62L(+) vv-specific CD8 T cells are bonafide memory cells. Our results are thus consistent with the branched differentiation model, where effector and memory cells develop simultaneously. These results are likely to have implications in the context of vaccine design, particularly those based on vaccinia virus recombinants.

Memory T cells are composed of effector, central, and memory stem cells. Previous studies have implicated that both T-bet and Eomes are involved in the generation of effector and central memory CD8 T cells. The exact role of these transcription factors in shaping the memory T cell pool is not well understood, particularly with memory stem T cells. Here, we demonstrate that both T-bet or Eomes are required for elimination of established tumors by adoptively transferred CD8 T cells. We also examined the role of T-bet and Eomes in the generation of tumor-specific memory T cell subsets upon adoptive transfer. We showed that combined T-bet and Eomes deficiency resulted in a severe reduction in the number of effector/central memory T cells but an increase in the percentage of CD62L(high)CD44(low) Sca-1(+) T cells which were similar to the phenotype of memory stem T cells. Despite preserving large numbers of phenotypic memory stem T cells, the lack of both of T-bet and Eomes resulted in a profound defect in antitumor memory responses, suggesting T-bet and Eomes are crucial for the antitumor function of these memory T cells. Our study establishes that T-bet and Eomes cooperate to promote the phenotype of effector/central memory CD8 T cell versus that of memory stem like T cells.

Most memory phenotype (MP) CD44(hi) CD8(+) cells are resting interleukin (IL)-15-dependent cells characterized by high expression of the IL-2/IL-15 receptor beta (CD122). However, some MP CD8(+) cells have a CD122(lo) phenotype and are IL-15 independent. Here, evidence is presented that the CD122(lo) subset of MP CD8(+) cells is controlled largely by major histocompatibility complex (MHC) class I molecules. Many of these cells display surface markers typical of recently activated T cells (CD62L(lo), CD69(hi), CD43(hi), and CD127(lo)) and show a high rate of background proliferation. Cells with this phenotype are highly enriched in common gamma chain-deficient mice and absent from MHC-I(-/-) mice. Unlike CD122(hi) CD8(+) cells, CD122(lo) MP CD8(+) cells survive poorly after transfer to MHC-I(-/-) hosts and cease to proliferate. Although distinctly different from typical antigen-specific memory cells, CD122(lo) MP CD8(+) cells closely resemble the antigen-dependent memory CD8(+) cells found in chronic viral infections.

We characterized T cell phenotypes in 74 paired blood and cerebrospinal fluid (CSF) samples of HIV-infected and uninfected persons using four-color flow cytometry. CD4+ and CD8+ T cells subsets were further characterized by identifying activated/resting and memory/naive subsets in CSF and blood using the markers CD38/HLA-DR and CD45RA/CD62L, respectively. With and without HIV-infection, the proportion of CD4+ T cells and memory T cells among T cells in CSF was higher compared to blood. In HIV-infection, activated CD4+ and CD8+ T cells in CSF were more abundant than in uninfected controls. As expected, combination antiretroviral therapy (ART) reduced T cell activation in CSF and blood.

Memory Thy-1(+)CD8(+) T cells specific for the influenza A virus nucleoprotein (NP(366-374)) peptide were sorted after staining with the D(b)NP(366) tetramer, labeled with CFSE, and transferred into normal Thy-1.2(+) recipients. The donor D(b)NP(366)(+) T cells recovered 2 days later from the spleens of the Thy-1.2(+) hosts showed the CD62L(low)CD44(high)CD69(low) phenotype, characteristic of the population analyzed before transfer, and were present at frequencies equivalent to those detected previously in mice primed once by a single exposure to an influenza A virus. Analysis of CFSE-staining profiles established that resting tetramer(+) T cells divided slowly over the next 30 days, while the numbers in the spleen decreased about 3-fold. Intranasal infection shortly after cell transfer with a noncross-reactive influenza B virus induced some of the donor D(b)NP(366)(+) T cells to cycle, but there was no increase in the total number of transferred cells. By contrast, comparable challenge with an influenza A virus caused substantial clonal expansion, and loss of the CFSE label. Unexpectedly, the recruitment of naive Thy-1.2(+)CD8(+)D(b)NP(366)(+) host D(b)NP(366)(+) T cells following influenza A challenge was not obviously diminished by the presence of the memory Thy-1.1(+)CD8(+)D(b)NP(366)(+) donor D(b)NP(366)(+) set. Furthermore, the splenic response to an epitope (D(b)PA(224)) derived from the influenza acid polymerase (PA(224-233)) was significantly enhanced in the mice given the donor D(b)NP(366)(+) memory population. These experiments indicate that an apparent recall response may be comprised of both naive and memory CD8(+) T cells.

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by the appearance of a major CD8 subpopulation with reduced expression of the CD8 beta chain (CD8alpha+betalo). CD8 antiviral activity was subsequently shown to be mediated by the CD8alpha+betalo phenotype, which is the dominant CD8 phenotype in long-term infected cats. Two- and three-color flow cytometric analysis demonstrated that the CD8alpha+betalo subset is L-selectin negative (CD62L-) and has increased expression of CD44, CD49d, and CD18, consistent with an activation phenotype. The CD8alpha+betaloCD62L- cells but not the CD8alpha+betahiCD62L+ cells demonstrated strong antiviral activity in the FIV acute-infection assay. The progressive expansion of the CD8alpha+betaloCD62L- effector subset cells in FIV-infected cats parallels that seen in human immunodeficiency virus (HIV)-infected patients, suggesting that failure in homeostatic mechanisms regulating lymphocyte activation or trafficking (or both) may be a consequence of both HIV and FIV infections.

Bacterial infections are frequent complications in patients with liver cirrhosis. Cirrhotic patients present abnormalities in both innate and adaptive immune responses, including a deficient neutrophil recruitment to infected sites. The purpose of this study was to assess neutrophil-endothelium interactions in cirrhotic patients and evaluate the effects of G-CSF on this process. We studied neutrophil adhesion and transendothelial migration in 14 cirrhotic patients and 14 healthy controls. We also analyzed neutrophil expression of the adhesion molecules CD62L and CD11b in whole blood by flow cytometry. Cirrhotic patients expressed higher levels of CD11b than healthy controls, whereas CD62L expression was significantly lower, suggesting exposure of neutrophils to activating agents within the bloodstream. Neutrophils from cirrhotic patients showed increased adhesion to both resting and tumor necrosis factor alpha-stimulated microvascular endothelial cells and decreased transendothelial migration. Granulocyte colony-stimulating factor (G-CSF) (100 ng/ml) significantly enhanced neutrophil adhesion to microvascular endothelial cells in healthy controls but not in cirrhotic patients. G-CSF also significantly improved neutrophil transmigration in cirrhotic patients and healthy controls. In conclusion, cirrhotic patients exhibit increased neutrophil adhesion to microvascular endothelium and deficient transendothelial migration. G-CSF enhances neutrophil transendothelial migration in cirrhotic patients despite having no effect on neutrophil adhesion. Therefore, G-CSF may be able to increase neutrophil recruitment into infected sites in these patients.

Behçet's uveitis, characterized by chronic recurrent uveitis and obliterating retinal vasculitis, frequently causes bilateral blindness. Intraocular infiltration of TCRalphabeta+CD8brightCD56+ cells was a distinct feature in Behçet's uveitis. However, phenotypic natures and effector functions of the cells have remained elusive. This study was conducted to determine phenotypic and functional characteristics and cytotoxic mechanisms of CD8brightCD56+ T cells in Behçet's uveitis. CD11b+CD27-CD62L- phenotypes of CD8brightCD56+ T cells were increased in patients with active Behçet's uveitis compared with inactive Behcet's patients and normal controls. Interestingly, CD45RAdimCD45RO- phenotypes were expanded, and CD94 expression was markedly up-regulated in contrast to the down-regulation of NKG2D. Furthermore, these subsets were polarized to produce IFN-gamma and contained high amounts of preformed intracellular perforin while exclusively expressing surface FasL upon PI stimulation. Moreover, the cytolytic functions of freshly isolated CD8brightCD56+ T cells were up-regulated against both K562 (NK-sensitive) and Raji (NK-resistant) cells, which were effectively inhibited by perforin inhibitor (concanamycin A). Their cytolytic activity against HUVECs was also increased and was effectively suppressed by Fas ligand inhibitor (brefeldin A) and partly by perforin inhibitor. Furthermore, cytolytic functions of PMA and ionomycin-stimulated CD8brightCD56+ T cells against HUVECs were greatly enhanced, by pretreatment of recombinant human IFN-gamma on HUVECs. Therefore, CD8brightCD56+ T cells in Behçet's uveitis are characterized by cytotoxic effector phenotypes with functional NK receptors and function as strong cytotoxic effectors through both Fas ligand-dependent and perforin-dependent pathways.

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.

OBJECTIVE

Preeclampsia and pregnancies complicated by small-for-gestational age (SGA) neonates share several underlying mechanisms of disease. However, while an exaggerated systemic maternal inflammatory response is regarded as one of the hallmarks of the pathogenesis of preeclampsia, the presence of a similar systemic intra-vascular inflammation in mothers of SGA neonates without hypertension is controversial. The aim of this study was to determine phenotypic and metabolic changes in granulocytes and monocytes of women who develop preeclampsia and those who deliver an SGA neonate, compared to normal pregnant women.

METHODS

This cross-sectional study included patients with a normal pregnancy (n = 33), preeclampsia (n = 33), and an SGA without preeclampsia (n = 33), matched for gestational age at blood sample collection. Granulocyte and monocyte phenotypes were determined by flow cytometry, using monoclonal antibodies against selective cluster of differentiation (CD) antigens. The panel of antibodies included the following: CD11b, CD14, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR. Intracellular reactive oxygen species (iROS) were assessed at the basal state and after stimulation (oxidative burst). Results were reported as mean channel brightness (MCB) or intensity of detected fluorescence. Analysis was conducted with non-parametric statistics. A p-value < 0.01 was considered statistically significant.

RESULTS

(1) Women who delivered an SGA neonate had a higher MCB of CD11b in granulocytes and monocytes than those with a normal pregnancy (p < 0.001 for both); (2) patients with preeclampsia had a lower median MCB of CD62L in granulocytes (p = 0.006) and a higher median basal iROS and oxidative burst in monocytes than women with an SGA neonate (p = 0.003 and p = 0.002, respectively).

CONCLUSIONS

Pregnancies complicated by the delivery of an SGA neonate are characterized by a higher activation of maternal peripheral leukocytes than in normal pregnancies, but lower than in pregnancies complicated by preeclampsia.

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8+ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA+ CD8+ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8+ CTLs from naive CD45RA+ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.

BACKGROUND

During the first line defense of an infected host, circulating neutrophils invade the inflamed tissue, whereas mature neutrophils from the bone marrow pool migrate into the blood circulation and from there reinforce tissue infiltration. The CXC chemokine CXCL8, also know as interleukin-8, is a potent attractant of neutrophils. Recently, we discovered a new natural post-translational modification of CXCL8, i.e. the deimination of arginine into citrulline by peptidylarginine deiminases.

METHODS

The ability to provoke leukocytosis was assessed by intravenous administration of citrullinated CXCL8 in rabbits. Adsorption of citrullinated CXCL8 to the Duffy antigen/receptor for chemokines on human or rabbit erythrocytes was evaluated using a competitive binding assay. Finally, surface expression of adhesion molecules was studied after stimulating neutrophils with citrullinated CXCL8.

RESULTS

Citrullination of CXCL8 significantly increased this chemokine's ability to recruit neutrophils into the blood circulation. In addition, the competitive binding properties of CXCL8 for the Duffy antigen/receptor for chemokines were impaired upon citrullination. Since the Duffy antigen/receptor for chemokines is an important scavenging receptor for CXCL8 in the blood stream, citrullination may delay CXCL8 clearance from the circulation. Furthermore, the shedding of CD62L (L-selectin) and the upregulation of CD11b (beta2-integrin) protein expression on CXCL8-induced neutrophils were improved by deimination of CXCL8, possibly contributing to the neutrophil egress from the bone marrow. Conversely, surface expression of CD15, the neutrophilic ligand of endothelial selectins, was equally well upregulated by intact and citrullinated CXCL8.

CONCLUSIONS

These data show that citrullination of CXCL8 enhances leukocytosis, possibly through impaired chemokine clearance from the blood circulation and prolonged presentation to the bone marrow.

Neutrophil (polymorphonuclear neutrophil) migration across epithelial barriers is a common morphologic feature of many diseases. Previous studies show that PMN-epithelial interactions are dependent on the PMN beta 2-integrin CD11b/18; however, nothing is known about surface carbohydrates and PMN-epithelial interactions. Here we investigate the role of carbohydrates on PMN-epithelial interactions using PMN and cultured monolayers of the intestinal epithelial cell line T84. Addition of the carbohydrates mannose 6-phosphate (Man-6-P) and glucose 6-phosphate (Glu-6-P), but not fructose 1-phosphate (Fru-1-P) inhibited transmigration by>> or = 70%. Likewise, more complex carbohydrates, such as fucoidin and the Man-6-P-rich polysaccharide PPME selectively inhibited PMN transepithelial migration. These carbohydrates were found to be inhibitory in the apical-to-basolateral direction as well as the basolateral-to-apical direction, indicating a lack of polarity. This panel of related carbohydrates, however, was not effective in modulating short-term adhesion of PMN to epithelial monolayers, indicating that carbohydrate ligands may modulate different steps in the transmigration cascade. Finally, addition of functionally inhibitory monoclonal antibodies specific for the selectins (CD62E, CD62L, and CD62P) revealed no observable effect on PMN transmigration. These studies suggest that cell surface carbohydrates may play a role in inflammatory processes of the intestine.

In the present study we have compared the immunophenotypic characteristics of the CD56+lo and CD56+hi NK-cell subsets in a group of normal healthy adults. Our results show that CD56+hi NK-cells display greater light-scatter properties than CD56+lo NK-cells at the same time they have higher levels of CD25 and CD122 IL-2 chains, together with a higher reactivity for HLA-DR and CD45RO and lower levels of CD45RA, supporting that, as opposed to the majority of the CD56+lo population, CD56+hi NK-cells might correspond to a subset of activated circulating NK-lymphocytes. Higher expression of the CD2 and CD7 costimulatory molecules found for the CD56+hi NK-cells would support their greater ability to respond to various stimuli. In addition, CD56+hi NK-cells expressed higher levels of several adhesion molecules such as CD2, CD11c, CD44, CD56, and CD62L compared to CD56+lo NK-cells, supporting a particular ability of these cells to migrate from blood to tissues and/or a potential advantage to form conjugates with target cells. Interestingly, CD56+lo and CD56+hi NK-cells showed a different pattern of expression of killer receptors that might determine different activation requirements for each of these NK-cell subsets. For instance, absence or low levels of CD16 expression might explain the lower antibody-dependent cytotoxicity activity of CD56+hi NK-cells. On the other hand, the virtual absence of expression of the CD158a and NKB1 immunoglobulin-like and the greater reactivity for the CD94 lectin-like killer receptors on CD56+hi in comparison to CD56+lo NK-cells might determine different MHC-class I specificities for both NK-cell subsets, a possibility that deserves further studies to be confirmed.

Activation of murine T cells by Ag or mitogens results in changes in the expression of several cell-surface adhesion molecules that alter the way in which these cells migrate and localize in tissues in vivo. As naive CD8 precursor cells in lymph nodes (LN) differentiate into effector CTL in response to a skin allograft, they up-regulate their expression of Pgp-1 (CD44), VLA-4, and LFA-1 (CD11a/18), while down-regulating L-selectin (CD62L). All cytolytic activity is therefore present in this minor population of L-selectin- Pgp-1high LN T cells. We now report that, late after rejection of minor histocompatibility Ag-disparate skin grafts, the majority of memory CD8 T cells express both L-selectin and Pgp-1 and thus would be expected to migrate via the classical route of recirculation through LN. Furthermore, restimulation of these memory cells by Ag causes them to down-regulate L-selectin, so that memory-effector cells have the same L-selectin- Pgp-1high phenotype as do primary effector cells. These results are in contrast to recent reports that murine and ovine CD4 memory cells do not express L-selectin or recirculate through LN high endothelial venules. In addition, although virgin and naive CD4 cells may be divided based upon their expression of CD45RA or CD45RB, memory CD8 cells cannot be differentiated by their expression of CD45 isoforms.

ICAM-1/LFA-1 interactions are known to enhance T cell/APC interactions and to promote T cell activation and cytokine secretion. We have analyzed the consequences of ICAM-1-mediated signaling on the generation of memory T cell subsets. We report that lack of ICAM-1 on APCs, but not on T cells, leads to poor T cell activation and proliferation in vitro and in vivo, and that the defect can be compensated by Ag dose, exogenous IL-2, additional costimulation, and by increasing responder T cell density on APCs. ICAM-1-null mice do not respond to immunization with OVA peptide, but immunization with OVA or with Salmonella typhimurium leads to good T cell proliferation 7-10 days later, and clearance of a challenge infection is equivalent to that of wild-type mice. However, when followed over time, recall proliferation and antibacterial immunity decay rapidly in ICAM-1-null mice, while recall cytokine responses are unaffected. The decline in immunity is not related to poor survival of T cells activated on ICAM-1-null APCs, or to poor generation of effectors in ICAM-1-null mice. Phenotypic analysis of T cells stimulated on ICAM-1-null APCs reveals preferential generation of CD44(high) CD62L(low) effector memory cells (T(EM)) over CD44(high) CD62L(high) central memory cells (T(CM)). Further, while the proportion of naive:memory T cells is similar in unmanipulated wild-type and ICAM-1-null mice, there is an accumulation of T(EM) cells, and a high T(EM):T(CM) ratio in aging ICAM-1-null mice. Together, the data indicate that signaling through LFA-1 during T cell activation may be involved in commitment to a proliferation-competent memory pool.

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always>> 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Expression of the lymph node homing and CC-chemokine receptor 7 (CCR7), with L-selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T-cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62Lhigh CD44low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7high CD62Lhigh CD44high (central memory) and CCR7low CD62Llow CD44high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short-term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7high CD4 T cells produced predominantly interleukin (IL)-2, whereas CCR7low CD4 T cells produced both IL-2 and interferon-gamma (IFN-gamma). However, in contrast to previously published reports, the CCR7high CD8 T-cell subpopulation produced both IFN-gamma and IL-2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down-regulated CCR7 only after multiple cell divisions, and this coincided with the down-regulation of CD62L and production of IL-4 and IFN-gamma. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL-2- and IFN-gamma-producing cells are CCR7low, while few cytokine-expressing CCR7high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T-cell subpopulations, but indicate additional complexity in that CCR7high CD8 T cells also may produce IFN-gamma.

Strains of human immunodeficiency virus (HIV) transmitted between individuals use the CCR5 coreceptor, but no preferential depletion of particular Th-lymphocyte subpopulations has been reported during primary HIV infection (PHI). In contrast, gut-associated Th lymphocytes are preferentially depleted in macaques recently infected by simian immunodeficiency virus. The expression of CCR5 and the intestinal homing receptor integrin alpha4beta7 on subpopulations of Th lymphocytes was studied in 12 patients with PHI. There was a profound decrease of circulating alpha4beta7+ Th lymphocytes and CCR5+ memory Th lymphocytes with nonlymphoid homing potential (CD62L-CD45RO+). Unlike other Th lymphocytes, this cell population remained depleted despite early control of viral replication under antiretroviral treatment. Therefore, HIV preferentially targets a specific CCR5+ subpopulation of Th lymphocytes early during infection, inducing its persistent depletion despite treatment. Protective immunity in vivo depends on Th lymphocytes carrying homing capacity to nonlymphoid tissue, and therefore these data may explain the persistent abnormalities of immune functions in patients infected with HIV.

The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+) stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim) peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim) than CD56(bright) peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+)NKG2A(+) NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies.

Senescent T-lymphocytes are antigen-experienced cells that express the killer-cell lectin-like receptor G1 (KLRG1) and/or CD57; fail to clonally expand following further antigenic stimulation and prevail in the resting blood of older adults compared to the young. Physical exercise mobilises T-lymphocytes into the bloodstream and is therefore a model with which to compare age-related phenotypes of blood-resident T-cells with those T-cells entering the blood from peripheral lymphoid compartments. Eight young (Y; Age: 21+/-3 years) and 8 older (O; Age: 56+/-3 years) healthy males completed a maximal treadmill exercise protocol. Blood lymphocytes isolated before, immediately after and 1h after exercise were assessed for cell surface expression of KLRG1, CD57, CD28, CD45RA, CD45RO, CD62L and lymphocyte subset markers using three-colour flow cytometry. Lymphocyte subset numbers (CD3+, CD3+/CD4+, CD3+/CD8 and CD3-/CD56+) increased with exercise (p<0.05) but were not different between Y and O. At rest and immediately after exercise, the percentage of CD3+/CD8+ T-lymphocytes expressing KLRG1 and CD45RO was greater in O than Y, whereas Y had a greater expression of CD45RA and CD62L than O. The percentage of all CD3+/CD8+ and CD3+/CD4+ T-lymphocytes expressing KLRG1 and CD57 increased after exercise, but the magnitude of change was not age-dependent. In conclusion, there is a greater proportion of senescent CD3+/CD8+ T-lymphocytes in the blood of older adults compared to young at rest and immediately after exhaustive exercise, indicating that the greater frequency of KLRG1+/CD8+ T-lymphocytes in older humans is ubiquitous and not localised to the peripheral blood.

CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils.