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T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.

Animals↗

Pancreatic beta-cell-selective production of tumor necrosis factor-alpha induced by interleukin-1.

Cytokines have been regarded as effector molecules responsible for beta-cell death and major histocompatibility complex hyperexpression in endocrine pancreas of type I diabetes. However, the mechanism that results in beta-cell-selective destruction has not been elucidated. We demonstrated in this study, using cell lines of transformed mouse beta-cells and alpha-cells, that only pancreatic beta-cells but not alpha-cells produced tumor necrosis factor-alpha when exposed to interleukin-1 beta. Northern blot analysis confirmed the beta-cell-selective expression of tumor necrosis factor-alpha mRNA. Interleukin-1 beta also provoked tumor necrosis factor-alpha mRNA expression in vitro by normal mouse islet cells. Because tumor necrosis factor-alpha has been shown to potentiate beta-cell cytotoxicity of interleukin-1 and interferon-gamma, tumor necrosis factor-alpha produced in situ by beta-cells might be self-destructive. In fact, a low dose of interleukin-1 beta in combination with a low dose of interferon-gamma preferentially injured beta-cells. Hence endogenous tumor necrosis factor-alpha production by beta-cells may be involved in beta-cell-selective destruction in type 1 diabetes.

Animals↗

Polyadenylated RNA sequences from vaccinia virus-infected cells selectively inhibit translation in a cell-free system: structural properties and mechanism of inhibition.

The mechanism of vaccinia virus-induced selective inhibition of host cell protein synthesis was studied in a nonpermissive (Chinese hamster ovary, CHO) and in a permissive mouse cell line ( L cells). Small polyadenylated RNAs obtained from uninfected and infected cells were fractionated into six size classes by polyacrylamide gel electrophoresis. The RNAs from the first two largest fractions (greater than 500 nucleotide, nt) were translated into some low-molecular-weight polypeptides, whereas, the RNAs from the remaining fractions (400-500, 300-400, 200-300, and 100-200 nt) had no translational activity in reticulocyte lysates. When these nontranslating polyadenylated short sequences (POLADS) were added to the cell-free system together with HeLa cell mRNAs, translation was inhibited from 70%, by the 400- to 500-nt fraction, to about 20%, by the 100- to 200-nt fraction. The degree of inhibition of protein synthesis was clearly dependent on the size of POLADS. The translation of vaccinia virus mRNAs in the cell-free system was inhibited by about 25% with the 400- to 500-nt fraction, by 5% with the 300- to 400-nt fraction, while the smaller size POLADS had no inhibitory effect. The inhibition of HeLa cell and vaccinia virus mRNA translation by POLADS was reversed by the simultaneous addition of oligo(dT) to the cell-free system. POLADS were also obtained from uninfected cells, but they inhibited the translation of HeLa cell and vaccinia virus mRNAs to a much lesser extent. The removal of the poly(A) moiety from POLADS by treatment with ribonuclease H and oligo(dT) abolished their inhibitory effect on HeLa cell mRNA translation. The average length of the poly(A) tails of POLADS obtained from infected cells was longer than that of POLADS from normal cells. Inhibition of HeLa cell mRNA translation mediated by POLADS in the cell-free system was reversed (approximately 70%) by addition of crude initiation factors (ribosomal salt wash, RSW). Significantly, inhibition of translation of POLADS was reversed (greater than 90%) by addition of purified poly(A) binding protein (PAB). Purified initiation factor 4A (eIF-4A) also reversed this inhibition, but to a lesser extent than RSW and PAB. Our results show that the translation of vaccinia virus mRNAs is resistant to POLADS, suggesting that POLADS, by virtue of their long poly(A) tails, may sequester PAB and thus, play a role in selective inhibition.

Animals↗

Maintenance of pluripotential embryonic stem cells by stem cell selection.

As gastrulation proceeds, pluripotential stem cells with the capacity to contribute to all primary germ layers disappear from the mammalian embryo. The extinction of pluripotency also occurs during the formation of embryoid bodies from embryonic stem (ES) cells. In this report we show that if the initial differentiated progeny are removed from ES cell aggregates, further differentiation does not proceed and the stem cell population persists and expands. Significantly, the presence of even minor populations of differentiated cells lead to the complete loss of stem cells from the cultures. This finding implies that the normal elimination of pluripotent cells is dictated by inductive signals provided by differentiated progeny. We have exploited this observation to develop a strategy for the isolation of pluripotential cells. This approach, termed stem cell selection, may have widespread applicability to the derivation and propagation of stem cells.

Animals↗

Non-specific cell binding characteristics of para-magnetic polystyrene microspheres used for antibody-mediated cell selection.

The binding of cells to paramagnetic polystyrene microbeads in the absence of coupling antibodies was measured. Cells from normal bone marrow, from an acute lymphoblastic leukemia (ALL) cell line or from a neuroblastoma cell line, were labeled with the fluorochrome Hoechst 33342 and incubated with microbeads by rotation at 4 degrees C. Following this incubation, the microbeads with all attached cells were collected using an externally applied magnetic field and visualized by microscopic examination under ultraviolet illumination. The incubation variables included the protein content of the medium, and the period of rotation. Normal bone marrow was found to adhere sparingly to the microbeads; less than 1.0% of the total nucleated cell population was recovered with the beads, whereas greater than 5% of the ALL cells and greater than 30% of the neuroblastoma cells were found to bind non-specifically to the microspheres. Neither changes in the protein concentration of the medium or in the incubation period significantly altered the non-specific binding of the cell types examined. It is thus apparent that the use of these microspheres for positive selection of cells, such as the collection of hematopoietic stem cells for transplantation, would be compromised by a sizeable non-specific interaction. Modification of the surface of the microspheres to substantially reduce this interaction will be necessary before efforts at positive selection using the magnetic microspheres can be fruitful.

Antibodies↗

An analysis of B cell selection mechanisms in germinal centers.

Affinity maturation of antibodies during immune responses is achieved by multiple rounds of somatic hypermutation and subsequent preferential selection of those B cells that express B cell receptors with improved binding characteristics for the antigen. The mechanism underlying B cell selection has not yet been defined. By employing an agent-based model, we show that for physiologically reasonable parameter values affinity maturation can be driven by competition for neither binding sites nor antigen--even in the presence of competing secreted antibodies. Within the tested mechanisms, only clonal competition for T cell help or a refractory time for the interaction of centrocytes with follicular dendritic cells is found to enable affinity maturation while generating the experimentally observed germinal centre characteristics and tolerating large variations in the initial antigen density.

Animals↗

Effects of two glycine residues in positions 13 and 17 of pleurocidin on structure and bacterial cell selectivity.

Pleurocidin (Ple), a 25-residue alpha-helical antimicrobial peptide, isolated from skin mucosa of the winter flounder, shows potent bacterial cell selectivity. In this study, the effect of two glycine residues in positions 13 and 17 of Ple on structure and bacterial cell selectivity was investigated by Gly-->Ala substitution. Ala-substitution (Gly(13, 17)-->Ala, Gly13-->Ala and Gly17-->Ala) in positions 13 and 17 of Ple did not induce a significant change in antibacterial activity, but increased hemolytic activity. Both Gly(13, 17)-->Ala and Gly17-->Ala substitution did not cause a remarkable change in alpha-helical content in SDS micelles, while Gly(13, 17)-->Ala substitution caused a drastic increase in alpha-helical content. These results suggest that the hinge region from Gly13 to Gly17 of Ple is assumed to provide its conformational flexibility and bacterial cell selectivity.

Amino Acid Sequence↗

Signalling thresholds and negative B-cell selection in acute lymphoblastic leukaemia.

B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.

Amino Acid Motifs↗

CD1d-mediated stimulation of natural killer T cells selectively activates hepatic natural killer cells to eliminate experimentally disseminated hepatoma cells in murine liver.

Since hepatocellular carcinomas (HCCs) develop from transformed hepatocytes, sometimes in a multicentrical manner, immunological deletion of such small intrahepatic regions should be an important strategy to prevent HCC development. The liver contains abundant innate cell lineages including natural killer (NK) cells and natural killer T (NKT) cells, the latter of which become activated in a CD1d-restricted manner by alpha-galactosylceramide (alpha-GalCer). In our study, we investigated the anti-tumor effect elicited by alpha-GalCer administration against transplanted hepatoma cells in the liver, in comparison with that in extrahepatic sites. alpha-GalCer administration completely suppressed the growth of BNL 1MEA.7R.1 (BNL) hepatoma cells disseminated in the liver of syngeneic BALB/c mouse but had no anti-tumor effect on subcutaneously implanted BNL cells. Hepatic NKT cells became rapidly activated after alpha-GalCer administration compared to splenic NKT cells and then disappeared. Hepatic NK cells substantially increased their population as well as up-regulated their cytotoxic activity against BNL cells, but NK cells in other tissues, including the spleen, blood and lymph node, did not. Anti-asialo GM1 antibody treatment, which depleted NK cells in vivo, resulted in hepatic tumor formation in alpha-GalCer-treated mice, indicating the critical involvement of NK cells in the alpha-GalCer-induced anti-tumor effect in the liver. In conclusion, our study demonstrates clear differences in NK cell activation and anti-tumor effect through stimulation of NKT cells by alpha-GalCer between the liver and extrahepatic tissues. Sequential activation of these innate cell lineages may be an attractive strategy for controlling micro-disseminated hepatoma cells in the liver.

Animals↗

Induction of selective cell death targeting on mature T-lymphocytes in rats by a novel immunosuppressant, FTY720.

A novel immunosuppressant, FTY720, induces a rapid and marked decrease of peripheral lymphocytes, and prolong allograft survival in rats. Its mechanism of action is mediated by apoptotic cell death. In this study, we determined the time-related changes in the numbers of total lymphocytes, and the ratios of lymphocyte subpopulations in peripheral blood and lymphomyeloid organs in rats after the single oral administration of FTY720 (10 mg/kg), comparing with the effects of cyclophosphamide (80 mg/kg, ip). Total number of peripheral lymphocytes decreased significantly 3 h after the administration of the drug, while that of polymorphonuclear cells increased. T-cells were markedly decreased in number and reached a minimum of 2.3% of the control 3 days after the treatment, while B-cells reached 19.7%. T-cells decreased in spleen and liver but there was no notable change in thymus, lymph nodes, and bone marrow. The susceptibility of the cells against the drug was variant based on the type and the source of cells in vitro. Polymorphonuclear cells were the most resistant and lymph node cells the most sensitive to FTY720 after 3 h incubation with different concentration of the drug (1, 10,100 mumol/l). When incubated with 10 mumol/l of FTY720, B-cells were significantly higher in viability than the whole T- or CD4-cells. These results demonstrated that FTY720 induces cell death selectively in mature T-lymphocytes, especially CD4-lymphocytes, in peripheral blood without the depression of bone marrow.

Animals↗

BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex.

The tumor necrosis factor (TNF)-like ligand BAFF/BLyS (B-cell activating factor of the TNF family/B-lymphocyte stimulator) is a potent B-cell survival factor, yet its functional relationship with other B-cell surface molecules such as CD19 and CD40 is poorly understood. We found that follicular dendritic cells (FDCs) in human lymph nodes expressed BAFF abundantly. BAFF up-regulated a B cell-specific transcription factor Pax5/BSAP (Pax5/B cell-specific activator protein) activity and its target CD19, a major component of the B-cell coreceptor complex, and synergistically enhanced CD19 phosphorylation by B-cell antigen receptor (BCR). BAFF further enhanced B-cell proliferation, immunoglobulin G (IgG) production, and reactivity to CD154 by BCR/CD19 coligation and interleukin-15 (IL-15). Our results suggest that BAFF may play an important role in FDC-B-cell interactions through the B-cell coreceptor complex and a possibly sequential link between the T cell-independent and -dependent B-cell responses in the germinal centers.

Antibodies, Monoclonal↗

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide.

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.

Amino Acid Sequence↗

The role of the central L- or D-Pro residue on structure and mode of action of a cell-selective alpha-helical IsCT-derived antimicrobial peptide.

IsCT-P (ILKKIWKPIKKLF-NH2) is a novel alpha-helical antimicrobial peptide with bacterial cell selectivity designed from a scorpion-derived peptide IsCT. To investigate the role of L- or D-Pro kink on the structure and the mode of action of a short alpha-helical antimicrobial peptide with bacterial cell selectivity, we synthesized IsCT-p, in which D-Pro is substituted for L-Pro8 of IsCT-P. CD spectra revealed that IsCT-P adopted a typical alpha-helical structure in various membrane-mimicking conditions, whereas IsCT-p showed a random structure. This result indicated that D-Pro in the central position of a short alpha-helical peptide provides more remarkable structural flexibility than L-Pro. Despite its higher antibacterial activity, IsCT-p was much less effective at inducing dye leakage in the negatively charged liposome mimicking bacterial membrane and induced no or little membrane potential depolarization of Staphylococcus aureus. Confocal laser scanning microscopy showed that IsCT-p penetrated the bacterial cell membrane and accumulated in the cytoplasm, whereas IsCT-P remained outside or on the cell membrane. These results suggested that the major target of IsCT-P and IsCT-p is the bacterial membranes and intracellular components, respectively. Collectively, our results demonstrated that the central D-Pro kink in alpha-helical antimicrobial peptides plays an important role in penetrating bacterial membrane as well as bacterial cell selectivity.

Antimicrobial Cationic Peptides↗

Large-scale isolation of CD34+ cells using the Amgen cell selection device results in high levels of purity and recovery.

The Amgen Cell Selection Device (ACSD) is a fully automated system based on the research scale magnetic-activated cell separation (MACS) system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for the selection of CD34+ cells. Leukapheresis products (LP) (n = 30) from normal donors mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) were selected with the ACSD to evaluate the performance of this system. The starting LP contained a median of 0.51% CD34+ cells (range 0.21%-1.54%) and a median WBC count of 3.0 x 10(10) (range 1-4.7 x 10(10) cells). After selection on the ACSD a mean purity of 91.5% +/- 0.6% CD34+ cells was obtained, with a median purity of 95.5% CD34+ cells. A median of 98 x 10(6) total CD34+ cells were recovered postselection, with a range of 31-323 x 10(6) cells collected from the LP. This represented a mean recovery of 81.7% +/- 6% of CD34+ cells and a median of 78% compared with starting CD34+ cell numbers in the LP. FACS analysis of the selected products demonstrated a 4-5 log depletion of T cell subsets, including CD3, CD4, CD8, and CD56 subsets. These data demonstrate the high performance obtained with the ACSD resulting in a final product of greater than 90% purity of CD34+ cells. CD34+ cells selected with the ACSD represent an ideal product for clinical applications, such as tumor cell purging, T cell depletion for allogeneic transplant, ex vivo expansion, and gene therapy.

Antigens, CD↗

Isolex 300i CD34-selected cells to support multiple cycles of high-dose therapy.

BACKGROUND: We have previously reported that repeated cycles of high-dose therapy (HDT), can be supported by unmanipulated autologous PBPC. Here we investigate whether purified CD34+ cells, obtained by immunomagnetic separation using the Isolex 300i device, can support such therapy. METHODS: Twenty-nine consecutive patients with metastatic breast cancer had PBPC mobilized and harvested following chemotherapy and G-CSF (10 microg/kg per day). Patients with > 4.0 x 10(6)/kg CD34+ cells in the apheresis product underwent CD34-selection using the Isolex 300i (v2.0) device. All cells collected were equally divided into three aliquots and cryopreserved. Patients who did not achieve this threshold had unmanipulated cells collected and stored. Patients subsequently received three cycles of HDT with paclitaxel (175 mg/m2), thiotepa (300 mg/m2) and either ifosfamide (10 g/m2) or cyclophosphamide (4 g/m2). It was intended for patients to receive CD34-selected cells to support each of the three cycles of HDT (i.e 1/3 for each cycle) and to compare hemopoietic recovery between patients receiving CD34-selected cells or unmanipulated cells. RESULTS: Thirteen of the 29 patients (45%) did not mobilize sufficient CD34+ cells to undergo CD34-selection. The remaining 16 patients underwent CD34-selection with a median purity of 84.3% (range: 16.3-96.1%) and yield of 34% (range: 1-60%). Fifteen of these patients proceeded to HDT and 42 of the planned 45 cycles were administered. Nine patients had all three HDT cycles supported by CD34-selected cells. The median number of CD34-selected cells (x 10(6)/kg) infused per cycle was 1.5 (range: 0.04-3.01). Three of the 15 patients required infusion of 'back-up' unmanipulated cells because of delayed neutrophil recovery. Of the 13 patients whose PBPCs did not undergo CD34+ cell selection, 11 proceeded to HDT with a median of 3.2 x 10(6)/kg (range: 2.0-4.4) unselected cells infused per cycle and 31 of 33 planned cycles were delivered. When hemopoietic recovery was compared between cycles of HDT supported by CD34-selected (n = 34) and unmanipulated cells (n = 31), there was a modest slowing in the patients receiving CD34-selected cells; time to ANC > 1.0 x 10(9)/L = 11 days versus 10 days (P = 0.0122) and platelets > 20 x 10(9)/L = 14 days versus 13 days (P = 0.0009). No difference in recovery to 50 x 10(9)/L was observed (P = 0.54). CONCLUSION: We have demonstrated that Isolex 300i CD34-selected cells are capable of supporting multiple cycles of HDT. However, we were unable to acquire sufficient CD34+ cells to perform this processing in 45% (13/29) of patients and further improvements in yield are required to overcome the modest delay in neutrophil and platelet recovery.

Adult↗

Regulation of CD1 function and NK1.1(+) T cell selection and maturation by cathepsin S.

NK1.1(+) T cells develop and function through interactions with cell surface CD1 complexes. In I-A(b) mice lacking the invariant chain (Ii) processing enzyme, cathepsin S, NK1.1(+) T cell selection and function are impaired. In vitro, thymic dendritic cells (DCs) from cathepsin S(-/-) mice exhibit defective presentation of the CD1-restricted antigen, alpha-galactosylceramide (alpha-GalCer). CD1 dysfunction is secondary to defective trafficking of CD1, which colocalizes with Ii fragments and accumulates within endocytic compartments of cathepsin S(-/-) DCs. I-A(k), cathepsin S(-/-) mice do not accumulate class II-associated Ii fragments and accordingly do not display CD1 abnormalities. Thus, function of CD1 is critically linked to processing of Ii, revealing MHC class II haplotype and cathepsin S activity as regulators of NK T cells.

Animals↗