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Biomedical subjects

L A Sherman

Publications and source records attributed to L A Sherman.

At least 19 recordsLinked to original sources
PubMed2002

Antigenic and immunologic characterization of an allogeneic colon carcinoma vaccine.

We report the immunological characterization of three colon carcinoma cell lines, COLO 205, SW620 and SW403, which we selected to combine with cytokine-secreting fibroblasts for the development of an allogeneic tumour cell vaccine. The cell lines expressed HLA-A2 as well as shared tumour-associated antigens (TAAs) representative of colon carcinomas: CEA, Ep-CAM, MUC1, HER2/neu and MAGE antigens. They did not secrete high levels of the immunosuppressive factors TGF-beta, IL-10 or prostaglandins. The lines presented TAAs in a manner recognized by immune effector cells, which was demonstrated by the lysis of SW620 by HLA-A2-restricted anti-p53 cytotoxic T lymphocytes (CTL). COLO 205 and SW620 were genetically modified to express the co-stimulatory molecule CD80 (B7.1), which increased the ability of the cells to stimulate CTL in vitro. CTL clones derived from HLA-A2+ peripheral blood mononuclear cells stimulated with the CD80-expressing lines lysed the stimulator cell and an HLA-A2+ colon cancer cell line, but did not lyse an isogeneic fibroblast line or an HLA-A2- colon cancer cell line. CTL clones derived from colon carcinoma patients immunized with an allogeneic vaccine containing these lines demonstrated killing of autologous tumour cells, the vaccine cell lines and other HLA-A2+ colon cancer cell lines, but not fibroblasts isogeneic to certain of the target cell lines. Our studies demonstrate that these colon carcinoma cell lines express shared TAAs that can induce CTLs which recognize and lyse other colon carcinoma cells, and support the continued clinical evaluation of the CD80 gene modified allogeneic colon cell/cytokine-secreting fibroblast carcinoma vaccine.

Antigen Presentation↗
PubMed2001

Phenotypic and functional analysis of CD8(+) T cells undergoing peripheral deletion in response to cross-presentation of self-antigen.

Not all T cells specific for autoantigens are eliminated in the thymus, and therefore alternate mechanisms are required to prevent potentially autoreactive T cells from developing into effectors. Adoptive transfer of CD8(+) T cells from influenza hemagglutinin-specific Clone 4 TCR transgenic mice into mice that express hemagluttinin in the pancreatic islets results in tolerance. This is preceded by activation of Clone 4 T cells that encounter antigen cross-presented in the draining lymph nodes of the pancreas. In this report we compare the phenotype, function, and costimulatory requirements of Clone 4 T cells activated by endogenous self-antigen, with Clone 4 T cells stimulated by influenza virus. The cells undergoing tolerance upregulate both CD69 and CD44, yet only partially downregulate CD62L, and do not express CD49d or CD25. Most importantly, they lack the ability to produce interferon-gamma in response to antigen and show no cytolytic activity. Clone 4 T cells disappear after several cycles of division, apparently without leaving the site of initial activation. Surprisingly, despite the fact that such stimulation occurs through recognition of antigen that is cross-presented by a professional antigen-presenting cell, we find this activation is not dependent on costimulation through CD28. These data demonstrate that the recognition by naive CD8(+) T cells of cross-presented self-antigen results in localized proliferation and deletion, without the production of effector cells.

Animals↗
PubMed2001

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.

Adoptive Transfer↗
PubMed2001

The manganese stabilizing protein (MSP) and the control of O2 evolution in the unicellular, diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142.

The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N2 fixation from photosynthesis. To better understand the processes by which photosynthesis is regulated, we have analyzed Photosystem (PS) II O2 evolution and the PSII lumenal proteins, especially the Mn stabilizing protein (MSP). We describe a procedure using glycine betaine to isolate photosynthetic membranes from Cyanothece sp. that have high rates of PSI and PSII activity. Analysis with these membranes demonstrated similar patterns of O2 evolution in vivo and in vitro, with a trough at the time of maximal N2 fixation and with a peak in the late light period. The pattern of PSI activity was also similar in vivo and in vitro. We cloned the genes for MSP (psbO) and the 12 kDa protein (psbU) and analyzed their transcriptional properties throughout the diurnal cycle. We suggest that the changes in PSII activity in Cyanothece sp. were due to conformational changes in a highly flexible MSP, a suggestion which can now be studied in a chimera. The Cyanothece sp. psbO gene has been transformed into Synechocystis sp. PCC 6803; MSP and O2 evolution in the resulting transformant had properties that were similar to those in Cyanothece sp., providing additional confirmation for the properties of Cyanothece sp. MSP.

Amino Acid Sequence↗
PubMed2001

CTLA-4 blockade enhances the CTL responses to the p53 self-tumor antigen.

p53 is an attractive target for cancer immunotherapy because it is overexpressed in a high proportion of many different types of tumors. However, it is also expressed in normal tissues and acts as a toleragen in vivo. Previously, detailed examination of the repertoire specific for the murine p53(261-269) epitope in conventional and p53-deficient mice demonstrated that because of expression of p53, the CD8(+) T cells that respond to this epitope express low-affinity TCRs. It has been reported that tolerance to tumor Ags can be broken by in vivo administration of anti-CTLA-4 mAb. With the goal of overriding tolerance and achieving optimal activation of p53-specific CTL, the current study has assessed the effect of anti-CTLA-4 mAb on the p53-specific repertoire. It was found that blockade of CTLA-4 engagement at the time of antigenic stimulation induced a vigorous amplification of the CTL responses to p53 as well as proportionate expansion of the memory T cell pool. This effect was dependent on the presence of CD4(+) T cell help and correlated with an enhancement of helper function. However, anti-CTLA-4 treatment did not enhance the avidity of the resultant p53-specific CTL populations and, therefore, could not reverse this important consequence of tolerance.

Abatacept↗
PubMed2001

Pattern of cyanophycin accumulation in nitrogen-fixing and non-nitrogen-fixing cyanobacteria.

The temporal and spatial accumulation of cyanophycin was studied in two unicellular strains of cyanobacteria, the diazotrophic Cyanothece sp. strain ATCC 51142 and the non-diazotrophic Synechocystis sp. strain PCC 6803. Biochemistry and electron microscopy were used to monitor the dynamics of cyanophycin accumulation under nitrogen-sufficient and nitrogen-deficient conditions. In Cyanothece sp. ATCC 51142 grown under 12 h light/12 h dark nitrogen-fixing conditions, cyanophycin was temporally regulated relative to nitrogenase activity and accumulated in granules after nitrogenase activity commenced. Cyanophycin granules reached a maximum after the peak of nitrogenase activity and eventually were utilized completely. Knock-out mutants were constructed in Synechocystis sp. PCC 6803 cphA and cphB genes to analyze the function of these genes and cyanophycin accumulation under nitrogen-deficient growth conditions. The mutants grew under such conditions, but needed to degrade phycobilisomes as a nitrogen reserve. Granules could be seen in some wild-type cells after treatment with chloramphenicol, but were never found in Delta cphA and Delta cphB mutants. These results led to the conclusion that cyanophycin is temporally and spatially regulated in nitrogen-fixing strains such as Cyanothece sp. ATCC 51142 and represents a key nitrogen reserve in these organisms. However, cyanophycin appeared to play a less important role in the non-diazotrophic unicellular strains and phycobilisomes appeared to be the main nitrogen reserve.

Bacterial Proteins↗
PubMed2001

The T-cell repertoire available for recognition of self-antigens.

Autoimmunity can be viewed as the price we pay in order to have a highly diverse TCR repertoire. This is not to say that every effort isn't made by the immune system to prevent autoimmune disease. During thymic development, and later in the periphery, the TCR repertoire is continuously purged of cells that are activated by self-antigens. Other lymphocytes are recruited to join a legion of T regulatory cells that assist in preventing 'friendly fire'. Recent studies continue to reveal the importance of the composition of the T cell repertoire in predisposing an individual to autoimmune disease.

Animals↗
PubMed2001

The role of Fas-FasL in CD8+ T-cell-mediated insulin-dependent diabetes mellitus (IDDM).

In the past few years a number of studies have evaluated the contributions of different cytolytic pathways in the autoimmune destruction of pancreatic beta cells, which results in insulin-dependent (type I) diabetes mellitus. Conflicting results continue to emerge regarding the role of Fas-mediated apoptosis in beta-cell destruction. This is likely to reflect differences inherent to the model systems under investigation, as well as the pleiotropic nature of the genes that are involved in cytotoxicity. Despite these complications, it may be possible to reconcile some of these apparently conflicting results by considering that T-cell-mediated cytotoxicity can occur simultaneously by several mechanisms and that variables such as the cytokine milieu and the strength of the signal to the T cell received through the T-cell receptor complex may alter the relative contribution of each cytolytic pathway to beta-cell destruction.

Animals↗
PubMed2001

Identification and design of p53-derived HLA-A2-binding peptides with increased CTL immunogenicity.

The replacement of a suboptimal amino acid in a primary anchor position with an optimal residue improves human leucocyte antigen (HLA) binding and immunogenicity, while maintaining cytotoxic T lymphocyte (CTL) specificity. Using a neural network capable of performing quantitative predictions of peptide binding to HLA-A2 molecules, we identified three p53 protein-derived nonamer peptides with intermediate binding owing to suboptimal amino acids in the P2 anchor position. These peptides were synthesized along with the corresponding analogs, where the natural P2 residue had been replaced with the optimal leucine residue. All three modified peptides bound to and more efficiently stabilized HLA-A2 molecules than the corresponding nonmodified peptides. The HLA-A2 transgenic mice were used for immunization. Two of the epitopes were more immunogenic in their modified than in their natural versions. The CTLs raised against the modified peptides efficiently killed the target cells pulsed with the corresponding native peptide. In terms of sensitizing the targets cells for the CTL killing, the modified peptides were more efficient than native peptides. Finally, the CTLs induced by modified peptide killed HLA-A2 transgenic mouse fibrosarcoma cells transfected with human p53 DNA. The data suggest that modified self-peptides derived from overexpressed tumour-associated proteins can be used in vaccine development against cancer, and that quantitative predictions of HLA binding is of value in the rational selection and improvement of target epitopes recognized by CTLs.

Amino Acid Sequence↗
PubMed2001

Altered functional and biochemical response by CD8+ T cells that remain after tolerance.

To further define the molecular basis of tolerance to a peripherally expressed antigen we have correlated differences in functional capacity with biochemical events in hemagglutinin (HA)-specific cytotoxic T lymphocyte (CTL) clones derived either from a conventional B10.D2 mouse that is not tolerant to HA (D2 Clone 6) or from an InsHA mouse that is tolerant to HA (InsHA Clone 12). D2 Clone 6, but not InsHA Clone 12, triggers diabetes following in vivo transfer into irradiated InsHA hosts. This diabetogenic clone shows complete and sustained phosphorylation of TCR zeta chain and ZAP-70 following stimulation with HA-pulsed antigen-presenting cells. In contrast, InsHA Clone 12 showed only partial phosphorylation of TCR zeta and no phosphorylation of ZAP-70. There was no defect in activation or recruitment of Lck to the TCR complex in both the clones following stimulation with the cognate antigen. This deficiency in the proximal signaling in the InsHA Clone 12 could be overcome by increasing the strength of signal through the CD3-TCR complex, indicating that the signaling machinery of InsHA Clone 12 was functional. These data demonstrate that the HA-responsive CD8(+) T cells that can be retrieved from InsHA mice after tolerance induction respond to HA as a partial agonist/antagonist.

Animals↗
PubMed2000

Analysis of chlorophyll-protein complexes from the cyanobacterium Cyanothece sp. ATCC 51142 by non-denaturing gel electrophoresis.

The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N(2) fixation from photosynthesis. We are analyzing the mechanism by which photosynthesis is down-regulated so that O(2) evolution is minimized during N(2) fixation. Previous results suggested changes in photosynthesis that are mediated through the redox poise of the plastoquinone pool (a process involving state transitions, in which the redistribution of excitation energy between the two photosystems helps to optimize photosynthetic yield) and the oligomerization state of the photosystems. Our working hypothesis was that the regulation of photosynthesis involved changes in the oligomerization of the photosystems. To analyze this hypothesis, we utilized a low-ionic strength, non-denaturing gel electrophoresis system to study the Chl-protein complexes. We determined that PSI is mostly trimeric, whereas PSII appears mainly as monomers. We demonstrated that most of the Chl-protein complexes in Cyanothece sp. remained constant throughout the diurnal cycle, except for the transient accumulation of a Chl-protein complex (band C) which appeared only during the late light period. Based on the size of this complex, band C represents either an interaction of PSI and PSII or a PSII dimer. These results provide support for the dynamic nature of the photosystems with respect to the diurnal cycle.

Blotting, Western↗
PubMed2000

The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self tolerance on the TCR repertoire.

p53 is an attractive target for cancer immunotherapy since it is overexpressed in half of all tumors. However, it is also expressed in normal lymphoid tissue, and self tolerance leaves a p53-specific repertoire purged of high avidity CTL. To better understand the mechanism of tolerance and the basis for such low avidity interaction, p53-specific CTL from p53 deficient (p53-) and sufficient (p53+) A2.1/Kb transgenic mice were compared with respect to their ability to bind HLA-A2.1 tetramers containing cognate murine p53 peptide Ag, p53 261-269. Since the murine CD8 molecule cannot interact with human HLA-A2.1, this tests the ability of the TCR to bind the A2.1/peptide complex tetramer. CTL from p53- mice demonstrated strong binding of such A2.1/p53 261-269 tetramers; however, the CTL from tolerant p53+ mice were devoid of tetramer-binding CD8+ T cells. Examination of TCR expression at the clonal level revealed that CTL from p53+ and p53- mice each expressed comparable levels of the p53-specific TCR. These results indicate that normal expression of p53 promotes elimination of T cells expressing TCRs with sufficient affinity to achieve stable binding of the A2.1/p53 261-269 tetramers.

Amino Acid Sequence↗
PubMed2000

Characterization of CD8+ T lymphocytes that persist after peripheral tolerance to a self antigen expressed in the pancreas.

As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.

Animals↗
PubMed2000

Identification of iron-responsive, differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 with a customized amplification library.

We describe the use of a method called differential expression using customized amplification library (DECAL) to study the global changes in gene expression in iron-deficient versus iron-reconstituting cells of Synechocystis sp. strain PCC 6803. We identified a number of genes, such as isiA, idiA, psbA, cpcG, and slr0374, whose expression either increased or decreased in response to iron availability. Further analysis led to the identification of additional genes related to those identified by DECAL (e.g., psbC, psbO, psaA, apcABC, cpcBAC1C2D, and nblA) that were differentially regulated by iron availability. Expression of cpcG, psbC, psbO, psaA, apcABC, and cpcBAC1C2D increased, whereas that of isiA, idiA, nblA, psbA, and slr0374 decreased, in iron-reconstituting cells. S1 nuclease protection studies showed that increased transcript levels of psbA in iron-deficient cells was due to the increased expression of both psbA2 and psbA3 genes, although the steady-state level of psbA2 remained higher than that of psbA3.

Culture Media↗