Interleukin-7 in the transition of bone marrow progenitors to the thymus.

Interleukin-7 (IL-7) is essential for the development of T cells in humans and mice where deficiencies in IL-7 signaling result in severe immunodeficiency. T cells require IL-7 at multiple points during development; however, it is unclear when IL-7 is first necessary. We observed that mice with impaired IL-7 signaling had a large reduction in the number of early thymic progenitors (ETPs) while mice that overexpress IL-7 had greatly increased numbers of ETPs. These results indicated that the development of ETPs is sensitive to IL-7. Bone marrow progenitors of ETP are present in normal numbers in mice with impaired IL-7 signaling (IL-7Rα449F) and were efficiently recruited to the thymus. Furthermore, ETPs and their progenitors from IL-7Rα449F mice did not undergo increased apoptosis and proliferate normally compared to WT cells. Mixed bone marrow chimeras demonstrated that IL-7 signaling has a cell-intrinsic role in ETP development but was not required for development of bone marrow progenitors. We have shown a novel role for IL-7 signaling in the development of ETPs that is distinct from classic mechanisms of IL-7 regulating survival and proliferation.

Sialoadhesin ligand expression identifies a subset of CD4+Foxp3- T cells with a distinct activation and glycosylation profile.

Sialoadhesin (Sn) is a sialic acid-binding Ig-like lectin expressed selectively on macrophage subsets. In a model of experimental autoimmune encephalomyelitis, Sn interacted with sialylated ligands expressed selectively on CD4(+)Foxp3(+) regulatory T cells (Tregs) and inhibited their proliferation. In this study, we examined the induction of Sn ligands (SnL) on all splenic CD4(+) T cells following in vitro activation. Most CD4(+) Tregs strongly upregulated SnL, whereas only a small subset of ~20% CD4(+)Foxp3(-) T cells (effector T cells [Teffs]) upregulated SnL. SnL(+) Teffs displayed higher levels of activation markers CD25 and CD69, exhibited increased proliferation, and produced higher amounts of IL-2 and IFN-γ than corresponding SnL(-) Teffs. Coculture of activated Teffs with Sn(+) macrophages or Sn(+) Chinese hamster ovary cells resulted in increased cell death, suggesting a regulatory role for Sn-SnL interactions. The key importance of α2,3-sialylation in SnL expression was demonstrated by increased binding of α2,3-linkage-specific Maackia amurensis lectin, increased expression of α2,3-sialyltransferase ST3GalVI, and loss of SnL following treatment with an α2,3-linkage-specific sialidase. The induction of SnL on activated CD4(+) T cells was dependent on N-glycan rather than O-glycan biosynthesis and independent of the mucin-like molecules CD43 and P-selectin glycoprotein ligand-1, previously implicated in Sn interactions. Induction of ligands on CD4(+)Foxp3(-) Teffs was also observed in vivo using the New Zealand Black × New Zealand White F1 murine model of spontaneous lupus and SnL levels on Teffs correlated strongly with the degree of proteinuria. Collectively, these data indicate that SnL is a novel marker of activated CD4(+) Teffs that are implicated in the pathogenesis of autoimmune diseases.

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions.

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.

PSGL-1 function in immunity and steady state homeostasis.

The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.

CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis.

The sialomucin CD43 is highly expressed on most hematopoietic cells. In this study, we show that the CD43 ectodomain is shed from murine granulocytes, mast cells, and T cells, but not from macrophages. To study the significance of CD43 shedding, we constructed 2 CD43/34 chimeras in which the CD43 membrane-proximal or transmembrane domain was swapped with the corresponding domain from CD34 that is not shed from cells. Viability of cells that normally shed CD43 was negatively affected when forced to express either of the 2 CD43/34 chimeras, but toxicity was reduced when cells coexpressed wild-type CD43. The CD43 cytoplasmic tail (CD43ct) was found to translocate into the nucleus, and inhibition of either its nuclear translocation or its release by gamma-secretase was proapoptotic. Involvement of CD43 in regulation of apoptosis is consistent with our findings that CD43ct was modified by small ubiquitin-like modifier-1 and was colocalized with promyelocytic nuclear bodies. CD43-deficient cells exhibited reduced levels of promyelocytic nuclear bodies and had increased sensitivity to apoptosis induced by growth factor withdrawal or T-regulatory cell suppression. Taken together, our data indicate an essential function of CD43 processing and nuclear localization of CD43ct in cell homeostasis and apoptosis.

Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface.

CD43 is a large sialylated glycoprotein found on the surface of haematopoietic cells and has been previously shown to be necessary for efficient macrophage binding and immunological responsiveness to Mycobacterium tuberculosis. Using capsular material from M. tuberculosis and recombinant CD43-Fc, we have employed affinity chromatography to show that Cpn60.2 (Hsp65, GroEL), and to a lesser extent DnaK (Hsp70), bind to CD43. Competitive inhibition using recombinant protein and polyclonal F(ab')(2) antibody-mediated epitope masking studies were used to evaluate M. tuberculosis binding to CD43(+/+) versus CD43(-/-) macrophages. Results showed that Cpn60.2, but not DnaK, acts as a CD43-dependent mycobacterial adhesin for macrophage binding. Assessment of the specific binding between Cpn60.2 and CD43 showed it to be saturable, with a comparatively weak affinity in the low micromolar range. We have also shown that the ability of Cpn60.2 to competitively inhibit M. tuberculosis binding to macrophages is shared by the Escherichia coli homologue, GroEL, but not by the mouse and human Hsp60 homologues. These findings add to a growing field of research that implicates molecular chaperones as having extracellular functions, including bacterial adherence to host cells. Thus, CD43 may act as a Pattern Recognition Receptor (PRR) for bacterial homologues of the 60 kDa molecular chaperone.

Impaired innate immune response and enhanced pathology during Citrobacter rodentium infection in mice lacking functional P-selectin.

The selectin family of adhesion molecules mediates recruitment of immune cells to sites of inflammation which is critical for host resistance against infection. To characterize the role of selectins in host defence against Citrobacter rodentium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E- and L-selectin were infected using a Citrobacter-induced colitis model. Infected mice lacking PSGL-1 or P-selectin showed a more pronounced morbidity associated with higher bacterial load, elevated IL-12 p70, TNF-alpha, IFN-gamma, MCP-1 and IL-6 production, more severe inflammation and surprisingly higher leucocyte infiltration in the guts than WT control. Recruitment of neutrophils and macrophages and caecal inflammation were drastically reduced in infected P-selectin knockout mice receiving blocking monoclonal antibodies to ICAM-1 or LFA-1, indicating that these adhesion molecules may compensate for the loss of selectins in leucocyte recruitment. Furthermore, the adaptive immune response in mice lacking PSGL-1 or P-selectin remained functional since these infected mice were capable of eradicating the bacteria and being protected upon re-challenge with C. rodentium. These data demonstrate a definitive phenotypic impairment of innate response in mice lacking PSGL-1 or P-selectin, and suggest that these adhesion molecules are important in host innate immune response against Citrobacter infection.

Lymphocytes in the peritoneum home to the omentum and are activated by resident dendritic cells.

The omentum is of interest in the context of obesity-related metabolic disease where adipose tissue exhibits inflammatory changes; however, the immunology of the omentum is underexplored. The greater omentum is draped from the stomach and consists predominantly of adipose tissue studded with lymphoreticular aggregations (milky spots) that distinguish it from other visceral adipose tissues. Milky spots are thought to contain and conduct leukocytes in transit from the blood to the peritoneal cavity, particularly during peritonitis. We show here that both B and T lymphocytes counterflow from the peritoneal cavity to the omentum in mice. Residence in the omentum was brief with a t(1/2) residence time of 6 h. Omentum access was pertussis toxin-sensitive, dependent on activation of the Rap1 GTPase, and on the integrin LFA-1. B cells and CD44(high) T cells accessed the omentum most efficiently, but homing of resting CD44(low) T cells was also observed. Omental tissue from normal healthy mice was found to contain CD8(-)CD11b(high)MHC class II(high)CD11c(high) dendritic cells that promoted the rapid activation of T cells entering the omentum and cross-presented soluble OVA or OVA acquired from either OVA-expressing Escherichia coli or OVA-pulsed spleen cells. We conclude that the omentum incorporates two key features of immunological sentinel function, actively supported lymphocyte traffic and dendritic cells, that reinforce a conceptual framework for function in stimulating adaptive immunity. These results extend basic understanding of omental and peritoneal cavity immunology and of how proinflammatory events occurring within the peritoneal cavity might affect adipocyte and hepatocyte metabolism.

Lack of functional P-selectin ligand exacerbates Salmonella serovar typhimurium infection.

The selectin family of adhesion molecules mediates the recruitment of immune cells to the site of inflammation, which is critical for host survival of infection. To characterize the role of selectins in host defense against Salmonella Typhimurium infection, wild-type (WT) mice and mice lacking P-selectin glycoprotein ligand-1 (PSGL-1), P-, E-, or L-selectin, or the glycosyltransferase C2GlcNAcT-I (core 2) were infected using a Salmonella acute gastroenteritis model. Mice were monitored for survival and assessed for intestinal inflammation at 1 and 4 days postinfection. Infected mice lacking core 2, PSGL-1, or P-selectin showed a more pronounced morbidity and a significantly higher mortality rate associated with higher bacterial load and proinflammatory cytokine production, including that of TNF-alpha, MCP-1, and IL-6, from the colons at 4 days postinfection as compared with WT control. Surprisingly, at 1 day postinfection, more severe inflammation and higher neutrophil infiltration were observed in the ceca of mice lacking core 2, PSGL-1, or P-selectin compared with WT control. Enhanced levels of alpha(4)beta(7)(+) and MAdCAM-1(+) cells were observed in the ceca of infected mice lacking core 2, PSGL-1, or P-selectin. Neutrophil recruitment, cecal inflammation, and mortality rates were dramatically reduced in infected P-selectin knockout mice receiving blocking mAb to alpha(4)beta(7) integrin, indicating that this alternative adhesion molecule may attempt to compensate for the loss of selectins in neutrophil recruitment. These results demonstrate a definitive phenotypic abnormality in mice lacking core 2, PSGL-1, or P-selectin, suggesting that the interaction of functional PSGL-1 with P-selectin is an important process in host defense against Salmonella infection.

The metalloprotease-disintegrin ADAM8 is essential for the development of experimental asthma.

RATIONALE: Expression of the metalloprotease ADAM8 is increased in patients with asthma, but the functional significance of elevated ADAM8 expression in the context of asthma pathogenesis remains elusive. OBJECTIVES: To study development of asthma in ADAM8-deficient mice. METHODS: Ovalbumin-induced asthma was studied in wild-type, ADAM8-deficient, and ADAM8-chimeric mice. Lung inflammation was assessed by histology, analysis of bronchoalveolar lavage, and airway hyperresponsiveness. MEASUREMENTS AND MAIN RESULTS: ADAM8-deficient mice are highly resistant to the development of ovalbumin-induced airway inflammation and hyperresponsiveness. ADAM8 expression was induced in both hematopoietic cells and the nonhematopoietic microenvironment after induction of asthma, and ADAM8 expression in both cell populations was required for the full manifestation of asthma. Interestingly, loss of ADAM8 on T cells alone was sufficient to significantly decrease the asthma response. The attenuated response was not due to an intrinsic defect in antigen presentation or cytokine production but reflected an impaired migration of T cells, eosinophils, CD11b(+) CD11c(-), and CD11c(+) cells from blood vessels to the lung and alveolar space, suggesting a general hematopoietic cell deficiency in the absence of ADAM8. CONCLUSIONS: The results show that ADAM8 plays a proinflammatory role in airway inflammation. The milder disease outcome in the absence of ADAM8 suggests that this protein might be an interesting new target in treatment of this, and potentially other, inflammatory diseases in which recruitment of inflammatory cells is an essential part of pathogenesis.

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis.

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.

A cell-extrinsic ligand acquired by activated T cells in lymph node can bridge L-selectin and P-selectin.

P-selectin expressed on activated endothelia and platelets supports recruitment of leukocytes expressing P-selectin ligand to sites of inflammation. While monitoring P-selectin ligand expression on activated CD8+ T cells in murine adoptive transfer models, we observed two distinct ligands on responding donor cells, the canonical cell-intrinsic P-selectin ligand PSGL-1 and a second undocumented P-selectin ligand we provisionally named PSL2. PSL2 is unusual among selectin ligands in that it is cell-extrinsic, loaded onto L-selectin expressed by activated T cells but not L-selectin on resting naïve CD8+ T cells. PSL2 display is highest on activated T cells responding in peripheral lymph nodes and low on T cells responding in spleen suggesting that the original source of PSL2 is high endothelial venules, cells known to produce L-selectin ligands. PSL2 is a ligand for both P-selectin and L-selectin and can physically bridge the two selectins. The L-selectin/PSL2 complex can mediate P-selectin-dependent adherence of activated T cells to immobilized P-selectin or to activated platelets, either independently or cooperatively with PSGL-1. PSL2's capacity to bridge between L-selectin on activated T cells and P-selectin reveals an undocumented and unanticipated activity of cell-extrinsic selectin ligands in mediating selectin-selectin connectivity. The timing and circumstances of PSL2 detection on T cells, together with its capacity to support adherence to P-selectin-bearing substrates, are consistent with P-selectin engagement of both PSGL1 and the L-selectin/PSL2 complex during T cell recruitment. Engagement of PSGL-1 and L-selectin/PSL2 would likely deliver distinct signals known to be relevant in this process.

Requirement for core 2 O-glycans for optimal resistance to helminth infection.

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4ß7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4(+) T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 ß1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.

Deficiency of the metalloproteinase-disintegrin ADAM8 is associated with thymic hyper-cellularity.

BACKGROUND: Thymopoiesis requires thymocyte-stroma interactions and proteases that promote cell migration by degrading extracellular matrix and releasing essential cytokines and chemokines. A role for several members of the A Disintegrin and Metalloprotease (ADAM) family in T cell development has been reported in the past. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present data indicating that the family member ADAM8 plays a role in thymic T cell development. We used qrtPCR on FACS sorted thymic subsets together with immunofluorescence to analyze thymic ADAM8 expression. We found that ADAM8 was expressed in murine thymic stromal cells and at lower levels in thymocytes where its expression increased as cell matured, suggesting involvement of ADAM8 in thymopoiesis. Further flow cytometry analysis revealed that ADAM8 deficient mice showed normal development and expansion of immature thymocyte subsets. There was however an intrathymic accumulation of single positive CD4 and CD8 T cells which was most noticeable in the late mature T cell subsets. Accumulation of single positive T cells coincided with changes in the thymic architecture manifest in a decreased cortex/medulla ratio and an increase in medullary epithelial cells as determined by histology and flow cytometry. The increase in single positive T cells was thymus-intrinsic, independent of progenitor homing to the thymus or thymic exit rate of mature T cells. Chemotaxis assays revealed that ADAM8 deficiency was associated with reduced migration of single positive thymocytes towards CCL21. CONCLUSIONS/SIGNIFICANCE: Our results show that ADAM8 is involved in T cell maturation in the medulla and suggest a role for this protease in fine-tuning maturation of thymocytes in the medulla. In contrast to ADAM10 and ADAM17 lack of ADAM8 appears to have a relatively minor impact on T cell development, which was unexpected given that maturation of thymocytes is dependent on proper localization and timing of migration.

Thymic progenitor homing and lymphocyte homeostasis are linked via S1P-controlled expression of thymic P-selectin/CCL25.

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified sphingosine-1-phosphate (S1P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S1P.

Interaction of the selectin ligand PSGL-1 with chemokines CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid organs.

P-selectin glycoprotein ligand 1 (PSGL-1) is central to the trafficking of immune effector cells to areas of inflammation through direct interactions with P-selectin, E-selectin and L-selectin. Here we show that PSGL-1 was also required for efficient homing of resting T cells to secondary lymphoid organs but functioned independently of selectin binding. PSGL-1 mediated an enhanced chemotactic T cell response to the secondary lymphoid organ chemokines CCL21 and CCL19 but not to CXCL12 or to inflammatory chemokines. Our data show involvement of PSGL-1 in facilitating the entry of T cells into secondary lymphoid organs, thereby demonstrating the bifunctional nature of this molecule.

CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.

Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

Inducing P-selectin ligand formation in CD8 T cells: IL-2 and IL-12 are active in vitro but not required in vivo.

In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2(null) CD8 T cells responding in doubly deficient IL-2(null)IL-12(null) or IL-2(null)IL-15(null) male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.

CD34 and CD43 inhibit mast cell adhesion and are required for optimal mast cell reconstitution.

CD34 is a cell-surface sialomucin expressed by hematopoietic stem cells (HSC), mast cells, and vascular endothelia. Despite its popularity as an HSC marker, the function of CD34 on hematopoietic cells remains enigmatic. Here, we have addressed this issue by examining the behavior of mutant mast cells lacking CD34, the related sialomucin, CD43, or both molecules. Loss of these molecules leads to a gene-dose-dependent increase in mast cell homotypic aggregation with CD34/CD43KOs > CD43KO > CD34KO > wild-type. Importantly, reexpression of CD34 or CD43 in these cells caused reversal of this phenotype. Furthermore, we find that loss of these sialomucins prevents mast cell repopulation and hematopoietic precursor reconstitution in vivo. Our data provide clear-cut evidence for a hematopoietic function for CD34 and suggest that it acts as a negative regulator of cell adhesion.

An alternate core 2 beta1,6-N-acetylglucosaminyltransferase selectively contributes to P-selectin ligand formation in activated CD8 T cells.

Core 2 beta1,6-N-acetylglucosaminyltransferase (C2GlcNAcT) synthesizes essential core 2 O-glycans on selectin ligands, which mediate cell-cell adhesion required for lymphocyte trafficking. Although gene-deletion studies have implicated C2GlcNAcT-I in controlling selectin ligand-mediated cell trafficking, little is known about the role of the two other core 2 isoenzymes, C2GlcNAcT-II and C2GlcNAcT-III. We show that C2GlcNAcT-I-independent P-selectin ligand formation occurs in activated C2GlcNAcT-I(null) CD8 T cells. These CD8 T cells were capable of rolling under shear flow on immobilized P-selectin in a P-selectin glycoprotein ligand 1-dependent manner. RT-PCR analysis identified significant levels of C2GlcNAcT-III RNA, identifying this enzyme as a possible source of core 2 enzyme activity. Up-regulation of P-selectin ligand correlated with altered cell surface binding of the core 2-sensitive mAb 1B11, indicating that CD43 and CD45 are also physiological targets for this alternate C2GlcNAcT enzyme. Furthermore, C2GlcNAcT-I-independent P-selectin ligand induction was observed in an in vivo model. HY(tg) CD8 T cells from C2GlcNAcT-I(null) donors transferred into male recipients expressed P-selectin ligand in response to male Ag, although at reduced levels compared with wild-type HY(tg) CD8 T cells. Our data demonstrate that multiple C2GlcNAcT enzymes can contribute to P-selectin ligand formation and may cooperate with C2GlcNAcT-I in the control of CD8 T cell trafficking.