Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells.

Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.

PAR2 Promotes Vaccine-Induced Protection Against Helicobacter Infection in Mice.

BACKGROUND & AIMS: Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS: Immune responses against Helicobacter infection were compared between vaccinated PAR2(-/-) and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS: Following vaccination, PAR2(-/-) mice did not have reductions in Helicobacter felis infection (RUT values were 0.01 ± 0.01 for WT mice and 0.11 ± 0.13 for PAR2(-/-) mice; P < .05). The vaccinated PAR2(-/-) mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83 ± 1.47 for WT mice and 4.86 ± 1.35 for PAR2(-/-) mice; P < .002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350 ± 436 pg/mL for PAR2(-/-) mice; P < .03) and reduced recruitment of CD4(+) IL-17(+) T cells into the gastric mucosa of PAR2(-/-) mice following bacterial challenge (3.7% ± 1.5% for WT mice and 2.6% ± 1.1% for PAR2(-/-) mice; P < .05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4(+) T cells compared with DCs from PAR2(-/-) mice (4298 ± 347 and 3230 ± 779; P < .04), indicating that PAR2(-/-) DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2(+/+) DCs into vaccinated PAR2(-/-) mice increased vaccine-induced protection (RUT values were 0.11 ± 0.10 and 0.26 ± 0.15 for injected and noninjected mice, respectively; P < .03). CONCLUSIONS: PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51.

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.

Blood lymphocyte subsets in rats with adjuvant arthritis

To determine the phenotype of peripheral blood lymphocytes during the time-course of adjuvant arthritis (AA) to detect alterations that could be involved in the pathogenesis of the arthritic process. METHODS--Phenotype analysis was performed on days 7, 14, 21, 28, 42, 56 and 70 after arthritis induction using monoclonal antibodies to CD5, CD4 and CD8 subsets, and flow cytometry. The proportion of activated lymphocytes and lymphocytes was also assessed with monoclonal antibodies to IL-2R (CD25), to Ia antigen and by polyclonal antibodies to rat Ig. RESULTS--Adjuvant arthritis produced leukocytosis with neutrophilia. Rats with AA showed a marked increase in the number of both CD4+ and CD8+ cells. The ratio CD4/CD8 decreased because the rise in CD8+ cells was more pronounced than the increase in CD4+ cells. Changes in lymphocyte counts showed two well-defined periods: the first, from day 14 to day 28, during which the inflammation of the joints reached a maximum and changes in lymphocyte subsets were more pronounced, that is, there was a threefold increase in CD8+ lymphocytes over normal counts, and the second, from day 42 to day 70, in which modified parameters improved considerably but remained different from controls. CONCLUSION--Alterations were detected in the phenotype of peripheral blood lymphocytes in AA, which provides an additional marker of disease activity.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.

Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.

Heparin attenuates TNF-alpha induced inflammatory response through a CD11b dependent mechanism

Background In addition to its anticoagulant properties, heparin has anti-inflammatory effects, the molecular and mechanistic bases of which are incompletely defined. AIMS The current studies were designed to test the hypothesis that heparin abrogates the expression or function of leucocyte-endothelial adherence molecules which are fundamental to the acute inflammatory response. Methods The effects of heparin on tumour necrosis factor alpha (TNF-¿) induced leucocyte rolling, adhesion, and migration as well as vascular permeability were assessed in rat mesenteric venules using intravital microscopy. Expression of adhesion molecules was quantitated using a double radiolabelled monoclonal antibody (mAb) binding technique in vivo (P-selectin, intercellular cell adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1)) or flow cytometry (CD11a, CD11b, and L-selectin). Ex vivo binding of heparin to neutrophils was assessed by flow cytometry. RESULTS TNF-alpha induced a significant increase in leucocyte rolling, adhesion, and migration, and vascular permeability, coincident with a significant increase in expression of P-selectin, ICAM-1, and VCAM-1. Ex vivo assessment of blood neutrophils showed significant upregulation of CD11a and CD11b and significant downregulation of L-selectin within five hours of TNF-¿ administration. Heparin pretreatment significantly attenuated leucocyte rolling, adhesion, and migration but did not affect expression of cell adhesion molecules or vascular permeability elicited by TNF-¿ administration. Binding of heparin was significantly increased on blood neutrophils obtained five hours after TNF-¿ administration. Preincubation with an anti-CD11b mAb but not with an anti-CD11a or anti-L-selectin antibody significantly diminished heparin binding ex vivo.

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay.

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

MHC class I-related chain A conjugated to antitumor antibodies can sensitize tumor cells to specific lysis by natural killer cells.

PURPOSE: As a first step for the development of a new cancer immunotherapy strategy, we evaluated whether antibody-mediated coating by MHC class I-related chain A (MICA) could sensitize tumor cells to lysis by natural killer (NK) cells. EXPERIMENTAL DESIGN: Recombinant MICA (rMICA) was chemically conjugated to Fab' fragments from monoclonal antibodies specific for tumor-associated antigens, such as carcinoembryonic antigen, HER2, or CD20. RESULTS: Flow cytometry analysis showed an efficient coating of MICA-negative human cancer cell lines with the Fab-rMICA conjugates. This was strictly dependent on the expression of the appropriate tumor-associated antigens in the target cells. Importantly, preincubation of the tumor cells with the appropriate Fab-rMICA conjugate resulted in NK cell-mediated tumor cell lysis. Antibody blocking of the NKG2D receptor in NK cells prevented conjugate-mediated tumor cell lysis. CONCLUSIONS: These results open the way to the development of immunotherapy strategies based on antibody-mediated targeting of MICA.

A Candidate HIV/AIDS Vaccine (MVA-B) Lacking Vaccinia Virus Gene C6L Enhances Memory HIV-1-Specific T-Cell Responses.

The vaccinia virus (VACV) C6 protein has sequence similarities with the poxvirus family Pox_A46, involved in regulation of host immune responses, but its role is unknown. Here, we have characterized the C6 protein and its effects in virus replication, innate immune sensing and immunogenicity in vivo. C6 is a 18.2 kDa protein, which is expressed early during virus infection and localizes to the cytoplasm of infected cells. Deletion of the C6L gene from the poxvirus vector MVA-B expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (MVA-B ΔC6L) had no effect on virus growth kinetics; therefore C6 protein is not essential for virus replication. The innate immune signals elicited by MVA-B ΔC6L in human macrophages and monocyte-derived dendritic cells (moDCs) are characterized by the up-regulation of the expression of IFN-β and IFN-α/β-inducible genes. In a DNA prime/MVA boost immunization protocol in mice, flow cytometry analysis revealed that MVA-B ΔC6L enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+) and CD8(+) T-cell memory immune responses, with most of the HIV-1 responses mediated by the CD8(+) T-cell compartment with an effector phenotype. Significantly, while MVA-B induced preferentially Env- and Gag-specific CD8(+) T-cell responses, MVA-B ΔC6L induced more Gag-Pol-Nef-specific CD8(+) T-cell responses. Furthermore, MVA-B ΔC6L enhanced the levels of antibodies against Env in comparison with MVA-B. These findings revealed that C6 can be considered as an immunomodulator and that deleting C6L gene in MVA-B confers an immunological benefit by enhancing IFN-β-dependent responses and increasing the magnitude and quality of the T-cell memory immune responses to HIV-1 antigens. Our observations are relevant for the improvement of MVA vectors as HIV-1 vaccines.

MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells.

Major histocompatibility complex (MHC) class II-restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type-specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.

Control of the proliferation of activated CD4+ T cells by connexins.

As expression of Cxs in cells of the immune system increases upon cellular activation, we investigated whether Cxs and especially CxHcs play a major role during T cell-mediated responses. In particular, we studied the expression of Cx43Hc following CD4(+) T cell stimulation using flow cytometry, real-time PCR, and Western blot analysis. We showed that expression of Cx43 and its phosphorylated isoforms increased in response to the engagement of CD3 and CD28. Cx43Hcs were found to be involved in sustaining proliferation of T cells, as assessed by cell cycle staining, thymidine incorporation assays, and CFSE analysis of cells exposed to mimetic peptide inhibitors of the plasma membrane Cx channels and antibodies generated to an extracellular region of Cx. The reduction of T cell proliferation mediated by Cx channel inhibitors suppressed cysteine uptake but not cytokine production. We conclude that upon antigen recognition, T cells require CxHc to sustain their clonal expansion.

The tumor antigens RHAMM and G250/CAIX are expressed in head and neck squamous cell carcinomas and elicit specific CD8+ T cell responses.

Despite advances in surgery, radio- and chemotherapy, therapeutic approaches for patients with head and neck squamous carcinoma (HNSCC) need to be improved. Immunotherapies eliciting tumor specific immune responses might constitute novel treatment options. We therefore investigated the expression and immunogenicity of two tumor-associated antigens (TAA) the receptor for hyaluronic acid mediated motility (RHAMM) and carboanhydrase IX (G250/CAIX) in HNSCC patients. Twenty-two HNSCC samples were examined for the expression of RHAMM and G250 by Western blotting and immunohistochemistry, 14/22 samples were tested for HLA-A2 expression by flow cytometry. For 8/22 samples single tumor-cell suspensions were generated, and mixed lymphocyte peptide cultures (MLPC) were performed to evaluate the frequencies of cytotoxic T cells specifically recognizing RHAMM and G250 using Tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays. RHAMM and G250 were expressed in 73 and 80% of the HNSCC samples at the protein level. A co-expression of both TAAs could be detected in 60% of the patients. In 4/8 HLA-A2+ patients, 0.06-0.13% of CD8+ effector T cells recognized Tetramers for RHAMM or G250 and secreted IFNgamma and granzyme B in ELISPOT assays. RHAMM and G250 are expressed at high frequency and high protein level in HNSCCs and are recognized by cytotoxic CD8+ effector T cells. Therefore both TAAs constitute interesting targets for T cell based immunotherapies for HNSCC.