Melanomas are characterized by high metastatic potential, with regional lymph node representing the most frequent site of early dissemination in this disease. These regional lymph nodes also represent the primary site for differentiation of natural killer (NK) cells. Although blood-derived NK cells can efficiently lyse melanoma cells isolated from metastatic lymph node (M-LN), there has been no study of the properties of the most disease-relevant NK cells isolated from M-LN in patients with melanoma. Here, we report that M-LN contains 0.5% to 11% of CD56(bright) NK cells among CD45(+) hematopoietic cells present and that this cell population surrounds tumor cell clusters in M-LN. This NK cell population was characterized by expression of CD62L, chemokine receptors, and high levels of natural cytotoxicity receptors (NCR), NK group 2 D (NKG2D), and DNAX accessory molecule 1 (DNAM-1). Expression of NCR-NKp30 and NKG2D correlated negatively with percentages of tumor cells in M-LN. Interestingly, M-LN contained a unique subset of mature CD56(bright)CD16(+) NK cells displaying coregulated expression of NCR and NKG2D activating receptors. Ex vivo analyses suggested that M-LN-derived NK cells were inactive but could be activated by appropriate cytokine signals [interleukin (IL)-2 or IL-15], and could lyse metastatic melanoma cells in a highly efficient manner compared with blood-derived NK cells. Taken together, the results offer evidence that adjuvant immunotherapy that targets NK cells in M-LN for activation may improve treatment of patients with sentinel lymph node-positive melanoma.

Three distinct subsets of antigen-experienced CD8(+) T cells have been identified so far: short-living effector T cells (T(EC)) and two long-living subsets, described as central (T(CM)) and effector memory (T(EM)) T cells. The lineage relationships of these subpopulations as well as their involvement in protection have not yet been conclusively determined. We recently described a novel marker combination (CD127 and CD62L) to identify all three major CD8(+) T cell subsets in mice infected with Listeria monocytogenes (L.m.). Extensive lineage relationship analyses on highly purified subpopulations after in vitro and in vivo stimulation demonstrated that T(CM) can develop into T(EM) or T(EC), whereas T(EM) can only progress to T(EC) cells. Short-living T(EC) never regained a T(EM) or T(CM) phenotype. These data strongly suggest a hierarchical and unidirectional order of developmental stages. In vivo priming protocols that preferentially induced one of the different CD8(+) T cell subsets demonstrated that predominance of T(EM) (CD40 stimulation) correlated best with effective protection against L.m., whereas generation of neither T(CM) (by immunization with heat-killed L.m.) nor T(EC) (by systemic co-administration of CpG during primary infection) conferred substantial long-term protective immunity. These findings have important implications for the design of more effective T cell-based vaccines.

Chemokines induce the directional migration of targeted populations of leukocytes during periods of inflammation. Moreover, these molecules also regulate T-cell activation and differentiation following antigenic stimulation. In the present study, the contributions of the CC chemokine ligand 3 (CCL3) to the differentiation and migration of effector T cells in response to viral infection of the central nervous system (CNS) were analyzed. CCL3(-/-) mice infected with mouse hepatitis virus exhibited a significant reduction of virus-specific CD8(+) T cells within the CNS, correlating with delayed viral clearance. Decreased infiltration of CD8(+) T cells into infected CCL3(-/-) mice was associated with enhanced accumulation of primed CD8(+) T cells in cervical lymph nodes. Although virus-specific CD8(+) T cells from CCL3(-/-) mice were CD44(high), they remained CD62L(high) and CD25(low), retained CCR7 expression, and contained limited transcripts of the proinflammatory chemokine receptors CCR5 and CXCR3 compared with virus-specific CD8(+) T cells from CCL3(+/+) mice. Furthermore, the absence of CCL3 impaired the cytokine production and cytolytic activity of CD8(+) T cells. In addition, macrophage accumulation within the CNS was significantly decreased in infected CCL3(-/-) mice, correlating with reduced demyelination. These results suggest that CCL3 not only mediates macrophage chemotaxis but also significantly enhances differentiation of primed CD8(+) T cells into effector cells and their release into circulation, thus potentiating effective migration to the site of infection.

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.

CD8(+) T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8(+) T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L(-)KLRG1(+) effector-memory CD8(+) T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8(+) T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4(+) T cells, its potential to be sustained pathogenic CD4(+) T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4(+)CD45RB(high) T cells or colitogenic lamina propria (LP) CD4(+) memory T cells into immunodeficient IL-7(+/+) x RAG-1(-/-) and IL-7(-/-) x RAG-1(-/-) mice. Although IL-7(+/+) x RAG-1(-/-) recipients transferred with CD4(+)CD45RB(high) splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7(-/-) x RAG-1(-/-) recipients did not. Furthermore, IL-7(-/-) x RAG-1(-/-), but not IL-7(+/+) x RAG-1(-/-), mice transferred with LP CD4(+)CD44(high)CD62L(-)IL-7Ralpha(high) effector-memory T cells (T(EM)) isolated from colitic CD4(+)CD45RB(high)-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4(+) T(EM) cells was observed in the in IL-7(-/-) x RAG-1(-/-) mice to a similar extent of those in IL-7(+/+) x RAG-1(-/-) mice, Bcl-2 expression was significantly down-modulated in the transferred CD4(+) T cells in IL-7(-/-) x RAG-1(-/-) mice compared with those in IL-7(+/+) x RAG-1(-/-) mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4(+) T(EM) cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.

Complete DiGeorge syndrome is a fatal congenital disorder characterized by athymia, hypoparathyroidism, and heart defects. Less than half of patients are 22q11 hemizygous. The goal of this study was to assess if immune suppression followed by postnatal thymus transplantation would lead to T-cell function in 6 infant patients who had host T cells at the time of transplantation. All infants had fewer than 50 recent thymic emigrants (CD3(+)CD45RA(+)CD62L(+)) per cubic millimeter (mm(3)) and all had some proliferative response to the mitogen phytohemagglutinin. Four infants had rash, lymphadenopathy, and oligoclonal populations of T cells in the periphery. Five of 6 patients are alive at the follow-up interval of 15 months to 30 months. The 5 surviving patients developed a mean of 983 host CD3(+) T cells/mm(3) (range, 536/mm(3)-1574/mm(3)), a mean of 437 recent thymic emigrants/mm(3) (range, 196/mm(3)-785/mm(3)), and normal proliferative responses to phytohemaglutinin (follow-up from day 376 to day 873). The TCR repertoire became polyclonal in patients who presented with oligoclonal T cells. All patients had thymopoiesis on allograft biopsy. Postnatal thymus transplantation after treatment with Thymoglobulin shows promise as therapy for infants with complete DiGeorge syndrome who have significant proliferative responses to mitogens or who develop rash, lymphadenopathy, and oligoclonal T cells.

We investigated the primary cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (rVSVs). The primary response to Env peaked 5 to 7 days after intraperitoneal vaccination, at which time 40% of CD8(+) cells were Env tetramer positive and activated (CD62L(Lo)). These freshly isolated cells actively lysed target cells pulsed with the p18-I10 peptide and secreted gamma interferon and tumor necrosis factor alpha after stimulation with the Env p18-I10 peptide. The primary response to Env elicited by rVSVs was sixfold higher than that elicited by recombinant vaccinia viruses (rVVs) at 5 days postvaccination. An intranasal route of vaccination with VSV-Env also elicited a strong primary response to Env. The primary immune response to Gag elicited by rVSV peaked 7 days after vaccination, at which time 3% of CD8(+) cells were Gag tetramer positive and CD62L(Lo) and functional by intracellular cytokine staining. This response was eightfold higher than that elicited by rVV expressing Gag. VSV-GagEnv, which expresses both Gag and Env from a single recombinant, also induced strong cytotoxic T-lymphocyte (CTL) responses to both Env and Gag. Our quantitative analyses illustrate the potency of the VSV vector system in CTL induction.

Lung injury in a variety of disease states is critically dependent on neutrophil-mediated inflammatory responses. Neutrophil recruitment to sites of infection or tissue damage requires co-ordinated regulation of neutrophil adhesion and activation status. We have examined the effects of treatment of human peripheral blood neutrophils with priming agents [lipopolysaccharide (LPS). tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF)] upon expression of CD11a. CD11b. CD11c. CD35 and CD62-1 and CD11b function to assess whether subtle regulation of neutrophil adhesion potential accompanies augmented formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production. We have found that there are differential effects of priming concentrations of these agents. For LPS. CD62L loss occurs in the absence of changes in CD11b, whereas for PAF. CD11b up-regulation occurs in the absence of detectable loss of CD62-L. However, for TNF-2, decreased expression of CD62-L occurs concomitantly with increased expression of CD11b. In addition, we have shown that priming agents augment CD11b functional activity in a manner that parallels the priming of the respiratory burst. Thus, priming agents may differentially regulate neutrophil adhesive capacity and data presented in this manuscript suggest that the increased effector cell function observed in primed cells may be associated with a distinct repertoire of potential adhesive interactions.

Multiple subsets of the bone marrow contain T cell precursors, but it remains unclear which is most likely to replenish the adult thymus. Therefore, RAG-1+ early lymphoid progenitors (RAG-1+ ELP), and CD62L/L-selectin+ progenitors (LSP), as well as common lymphoid progenitors from C57BL6-Thy1.1-RAG-1/GFP mouse bone marrow were directly compared in transplantation assays. The two c-Kit(high) populations vigorously regenerated the thymus and were superior to common lymphoid progenitors in magnitude and frequency of thymic reconstitution. Regeneration was much faster than the 22 days described for transplanted stem cells, and RAG-1+ ELP produced small numbers of lymphocytes within 13 days. As previously reported, LSP were biased to a T cell fate, but this was not the case for RAG-1+ ELP. Although RAG-1+ ELP and LSP had reduced myeloid potential, they were both effective progenitors for T lymphocytes and NK cells. The LSP subset overlapped with and included most RAG-1+ ELP and many RAG-1- TdT+ ELP. LSP and RAG-1+ ELP were both present in the peripheral circulation, but RAG-1+ ELP had no exact counterpart among immature thymocytes. The most primitive of thymocytes were similar to Lin- c-Kit(high) L-selectin+ TdT+ RAG-1- progenitors present in the marrow, suggesting that this population is normally important for sustaining the adult thymus.

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

BACKGROUND

Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles.

RESULTS

Microparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 μm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes.

CONCLUSIONS

We have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.

The goal of this study was to compare the ability of donor naive and alloantigen-primed effector memory T cells to induce graft-vs-host disease after bone marrow transplantation in MHC-mismatched irradiated host mice. Purified CD4(+) naive (CD62L(high)CD44(low)) T cells and CD4(+) effector memory (CD62L(low)CD44(high)) T cells obtained from unprimed donors and donors primed to host alloantigens, respectively, were injected into host mice, and the rapidity, severity, and pattern of tissue injury of graft-vs-host disease was assessed. Unexpectedly, the naive T cells induced a more acute and severe colitis than the primed memory cells. Whereas the naive T cells expressing CD62L and CCR7 lymph node homing receptors vigorously expanded in mesenteric lymph nodes and colon by day 6 after transplantation, the primed memory T cells without these receptors had 20- to 100-fold lower accumulation at this early time point. These differences were reflected in the significantly more rapid decline in survival and weight loss induced by naive T cells. The primed memory T cells had a greater capacity to induce chronic colitis and liver injury and secrete IL-2 and IFN-gamma in response to alloantigenic stimulation compared with memory T cells from unprimed donors. Nevertheless, the expected increase in potency as compared with naive T cells was not observed due to differences in the pattern and kinetics of tissue injury.

Recent findings indicate that mesenchymal stem cells (MSCs) may act as a regulator of Th17 cell differentiation, however, the underlying mechanism is still under debate. To investigate the underlying mechanisms of MSCs' regulatory effect, mouse bone marrow-derived MSCs were cocultured with mouse CD4(+)CD25(low)CD44(low)CD62L(high) T cells in vitro, and the proportion of induced Th17 cells, cytokines secretion, and transcription factors expression were examined by flow cytometry, enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and Western blotting. For the first time, our results showed that bone marrow-derived MSCs were able to inhibit Th17 cell differentiation via interleukin (IL)-10 secretion as the Th17 cell proportion was significantly regained when IL-10 was neutralized, or expression of IL-10 by bone marrow-derived MSCs was downregulated by RNA interference technique. Furthermore, IL-10 may suppress expression of Rorγt, the key transcription factor for Th17 cells, both by activating suppressor of cytokine signaling 3 through signal transducers and activators of transcription 5 phosphorylation, and decreasing signal transducers and activators of transcription 3 binding, which is at the promoter of Rorγt. Thus, our results demonstrate the inhibitory effect of MSCs on Th17 cells differentiation, and suggest increased IL-10 secretion might be the key factor.

L-selectin (CD62L) is a type-I transmembrane glycoprotein and cell adhesion molecule that is expressed on most circulating leukocytes. Since its identification in 1983, L-selectin has been extensively characterized as a tethering/rolling receptor. There is now mounting evidence in the literature to suggest that L-selectin plays a role in regulating monocyte protrusion during transendothelial migration (TEM). The N-terminal calcium-dependent (C-type) lectin domain of L-selectin interacts with numerous glycans, including sialyl Lewis X (sLe^x) for tethering/rolling and proteoglycans for TEM. Although the signals downstream of L-selectin-dependent adhesion are poorly understood, they will invariably involve the short 17 amino acid cytoplasmic tail. In this review we will detail the expression of L-selectin in different immune cell subsets, and its influence on cell behavior. We will list some of the diverse glycans known to support L-selectin-dependent adhesion, within luminal and abluminal regions of the vessel wall. We will describe how each domain within L-selectin contributes to adhesion, migration and signal transduction. A significant focus on the L-selectin cytoplasmic tail and its proposed contribution to signaling via the ezrin-radixin-moesin (ERM) family of proteins will be outlined. Finally, we will discuss how ectodomain shedding of L-selectin during monocyte TEM is essential for the establishment of front-back cell polarity, bestowing emigrated cells the capacity to chemotax toward sites of damage.

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell-like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell-like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell-like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma-bearing mice. These findings provide a rationale for clinical exploitation of ex vivo-generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

The microvasculature of the normal lung contains a pool of sequestered neutrophils, which is markedly enhanced in acute lung inflammation. Lung neutrophil sequestration is determined by the cells' deformability and adhesivity to capillary endothelium, and is a pre-requisite for emigration into the airspaces. We assessed the effect of several pro-inflammatory mediators associated with acute lung inflammation on these factors. Platelet-activating factor, IL-8 and formyl-Met-Leu-Phe (fMLP) induced a marked, but transient reduction in neutrophil deformability. Also, increased surface expression of the beta(2)-integrin and CD11b, and shedding of L-selectin (CD62L) was observed for these stimuli. TNF-alpha in contrast caused a small decrease in cell deformability only after 30 min, and shedding of L-selectin, but no change in CD11b levels. However, TNF-alpha-pretreatment markedly enhanced the fMLP response for cell deformability, CD11b expression and CD62L loss. Moreover, all pre-treatments were found to induce chemokinesis, and all except fMLP, enhanced fMLP-directed chemotaxis. We were able to demonstrate, using specific TNF-alpha receptor antagonists, that the TNF-alpha-induced changes in chemotaxis were mediated through the 55-kDa receptor. Also, inhibitors of the mitogen activated protein (MAP) kinase signaling pathway showed that the p38 MAP kinase pathway was involved for fMLP-directed chemotaxis of TNF-pretreated neutrophils, although activation of the extracellular signal-regulated kinase (ERK) pathway was also seen. These data demonstrate the differential role of pro-inflammatory mediators in controlling neutrophil sequestration and migration, which may orchestrate the severity of the inflammatory response in such respiratory diseases as chronic obstructive pulmonary disease and asthma.

Converging experimental evidence indicates that CD4(+) regulatory T cells control progression of autoimmune insulitis in nonobese diabetic (NOD) mice. Here, we studied the nature of these regulatory T cells and their mode of action in diabetes-prone NOD Rag(-/-) or severe combined immunodeficient (SCID) mice harboring a transgenic T cell receptor derived from the diabetogenic T cell clone BDC2.5. We first show that diabetes onset is prevented in such mice by infusion of polyclonal CD4(+) T cells expressing L-selectin (CD62L) but not prevented or only marginally prevented by CD4(+)CD25(+) T cells. Similarly, we found with a cotransfer model that CD4(+)CD62L(+) T cells but not CD4(+)CD25(+) T cells inhibited diabetes transfer into NOD SCID recipients by transgenic NOD BDC2.5 SCID cells. Unexpectedly, cotransfer of transgenic NOD BDC2.5 SCID cells and spleen cells from WT diabetic NOD mice did not induce diabetes, whereas each individual population did so. Data are presented arguing for the role of CD4(+)CD62L(+) T cells present within the polyclonal diabetogenic population in mediating this apparently paradoxical effect. Collectively, these data confirm the central role of CD4(+)CD62L(+) regulatory T cells in controlling disease onset in a well defined transgenic model of autoimmune diabetes and suggest the intervention of homeostatic mechanisms as part of their mode of action.

SopB is a virulence factor of Salmonella encoded by SPI-5. Salmonella sopB deletion mutants are impaired in their ability to cause local inflammatory responses and fluid secretion into the intestinal lumen and also can enhance the immunogenicity of a vectored antigen. In this study, we evaluated the effects on immunogenicity and the efficacy of a sopB deletion mutation on two Salmonella enterica serovar Typhimurium vaccine strains with different attenuating mutations expressing a highly antigenic alpha-helical region of the Streptococcus pneumoniae surface protein PspA from an Asd(+)-balanced lethal plasmid. After oral administration to mice, the two pairs of strains induced high levels of serum antibodies specific for PspA as well as to Salmonella antigens. The levels of antigen-specific serum immunoglobulin G (IgG) and mucosal IgA were higher in mice immunized with sopB mutants. Enzyme-linked immunospot assay results indicated that the spleen cells from mice immunized with a sopB mutant showed higher interleukin-4 and gamma interferon secretion levels than did the mice immunized with the isogenic sopB(+) strain. The sopB mutants also induced higher numbers of CD4(+) CD44(hi) CD62L(hi) and CD8(+) CD44(hi) CD62L(hi) central memory T cells. Eight weeks after primary oral immunization, mice were challenged with 100 50% lethal doses of virulent S. pneumoniae WU2. Immunization with either of the sopB mutant strains led to increased levels of protection compared to that with the isogenic sopB(+) parent. Together, these results demonstrate that the deletion of sopB leads to an overall enhancement of the immunogenicity and efficacy of recombinant attenuated Salmonella vaccine strains.

Single-cell analysis of endogenous, primary CD8(+) T cell responses to the influenza D(b)NP(366) and D(b)PA(224) epitopes indicates that prominent clonotypes bearing "public" or "shared" T cell receptors (TCRs) subset early into CD62L(hi) and CD62L(lo) populations. The CD62L(lo) effectors divide more and are rapidly eliminated during the contraction phase, whereas stable CD62L(hi) memory populations persist in the long-term. Reflecting the high frequency of small CD62L(hi) clones expressing "private" TCRs, the TCR diversity range per mouse is generally two times higher within the CD62L(hi)CD8(+)D(b)NP(366)(+) set (1.6 times higher for CD62L(hi)CD8(+)D(b)PA(224)(+)) from 8 to >180 days after antigen challenge. Memory CD8(+)CD62L(hi) T cell precursors thus segregate from the outset into populations expressing "best-fit" and "suboptimal" TCR characteristics, with this pattern being maintained stably thereafter. Hence, our analysis suggests that early establishment of influenza-specific memory within the CD8(+)CD62L(hi) subset preserves clonal diversity and prevents "overdominance" by a few public, or shared, clones.

OBJECTIVE

Umbilical cord blood (UCB) T cells are predominantly CD45RA(+), secrete less cytokines, and have diminished cytotoxicity compared to adult peripheral blood (PB). We hypothesized that the functional impairment of bulk UCB cells results from the relative dominance of immature lymphocyte subsets. In this study we established the physiologic ranges of lymphocyte subsets in UCB, and contrasted those with adult PB.

METHODS

Four-color FACS was utilized to characterize surface and intracellular protein expression on lymphocyte subsets from fresh unmanipulated UCB and adult PB.

RESULTS

We found that UCB contain significantly higher absolute numbers of T cells, NK cells, and B cells than adult PB (p<0.0001). UCB also contains more "naïve" cells not only among CD4(+) and CD8(+) T cells but also among B lymphocytes (p=0.003). Most UCB T cells are CD45RA(+)/CD62L(+) "recent thymic emigrants" with smaller TCRgammadelta (p<0.0001) and CD25(+) subsets (p=0.0068). Fewer UCB T cells display HLA-DR and CCR-5 activation markers (p<0.0001) while the CD8(+)/CD57(+)/CD28(-) "suppressor," CD8(+)/CD45RA(+)/CD27(-) "cytotoxic," and skin homing CLA(+) T-cell subsets are absent altogether. Compared with adult PB, more cord blood T cells progress through cell cycle (p<0.0001) and enter apoptosis (p=0.0003). Unlike in adult PB, the majority of proliferating Ki-67(+) T cells in UCB retain a CD45RA(+)/RO(-), CD69(-), CD25(-), HLA-DR(-) "resting" phenotype (p=0.0002).

CONCLUSIONS

Most T and B lymphocytes express a nai;ve phenotype in cord blood while "suppressor" and "cytotoxic" T-cell subsets are absent. Cycling UCB T cells retain a nai;ve immunophenotype that may represent homeostatic expansion rather than antigen-driven proliferation.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

NKT cells are a small subset of T lymphocytes which express an invariant V(alpha24JalphaQ TCR and recognize glycolipids presented by CD1d. In adults, NKT cells have a memory phenotype, frequently associated with oligoclonal expansion, express NK cell markers, and produce TO cytokines upon primary stimulation. Because of these features, NKT cells are regarded as lymphocytes of innate immunity. We investigated NKT cells from cord blood to see how these cells appear in the absence of exogenous stimuli. We found that NKT cells are present at comparable frequencies in cord blood and adult peripheral blood mononuclear cells and in both cases display a memory (CD45RO+CD62L-) phenotype. However, neonatal NKT cells differ from their adult counterparts by the following characteristics: (1) they express markers of activation, such as CD25; (2) they are polyclonal; (3) they do not produce cytokines in response to primary stimulation. Together, our data show that human NKT cells arise in the newborn with an activated memory phenotype, probably due to recognition of an endogenous ligand(s). The absence of oligoclonal expansion and primary effector functions also suggest that neonatal NKT cells, despite their activated memory phenotype, require a further priming/differentiation event to behave as fully functional cells of innate immunity.