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S I Abrams

Publications and source records attributed to S I Abrams.

33 records · Page 2Linked to original sources
PubMed1996

Identification of overlapping epitopes in mutant ras oncogene peptides that activate CD4+ and CD8+ T cell responses.

Mutant ras p21 proteins contain sequences which distinguish them from normal endogenous ras and, thus, may represent unique epitopes for T cell recognition of antigen bearing tumor cells. Here, we examined the capacity of a mutant K-ras 9-mer peptide to induce in vivo CD8+ cytotoxic T lymphocytes (CTL). The peptide chosen reflected positions 4-12 of the point-mutated sequence of the K-ras oncogene encoding the Gly to Val substitution at codon 12. The overall rationale for selecting this particular 9-mer sequence was threefold: the mutant peptide contained a putative major histocompatibility complex (MHC) class I consensus anchor motif for murine H-2Kd; specific binding to MHC class I may then create an immunogenic complex for the induction of anti-ras CD8+ CTL; and finally, the mutant sequence overlapped with a newly characterized anti-ras CD4+ T helper type 1 epitope, which may have implications for the coordination and activation of both anti-ras immune mechanisms against the same target cell antigenic determinant. A functional interaction with H-2Kd was demonstrated with the mutant ras4-12(V12) peptide, but not the normal ras4-12(G12) peptide, which specifically inhibited an H-2Kd-restricted, anti-nucleoprotein NP147-155 CTL response in a dose-dependent fashion. An anti-ras CD8+ T cell line was then established from immune splenocytes of BALB/c (H-2d) mice injected with ras4-12 (V12) in adjuvant, which mediated peptide-specific lysis of syngeneic P815 tumor targets. Cytotoxicity was restricted by H-2Kd and strongly specific for the mutant ras peptide. Importantly, these anti-ras CTL specifically lysed a syngeneic tumor line (i.e. A20 lymphoma) transduced with the corresponding point-mutated ras oncogene, suggesting T cell receptor recognition of endogenously derived antigen. Overall, these data demonstrated that mutant ras p21 at codon 12(Gly-->Val) contained a peptide sequence which exhibited specific functional binding to a murine MHC class I molecule; the ability of the mutant, but not the normal sequence to bind selectively to murine MHC class I likely reflected the generation of a C-terminal anchor residue; and the ras4-12(V12) peptide was immunogenic for the production of antigen-specific CD8+ CTL, which lysed in vitro a syngeneic tumor cell line harboring the mutant K-ras oncogene.

Amino Acid Sequence↗
PubMed1996

Mutant ras epitopes as targets for cancer vaccines.

The ras p21 proto-oncogenes (ie, K-ras, H-ras, N-ras) encode a family of proteins vital to cellular signaling and function. Point mutations in these genes have been found in a wide diversity of human cancers, suggesting a strong association in the development of the malignant phenotype. Although the precise mechanisms leading to tumorigenesis are not fully understood, it has been proposed that point mutations in the ras p21 proto-oncogenes contribute to the transformation process through constitutive transduction of growth-promoting signals. These oncoproteins are distinct from normal ras p21 in both DNA and protein sequences at specific sites, typically positions 12, 13, 59, or 61. A large frequency of human cancers harbor point mutations in the ras gene at codon 12, where the normal Gly residue is substituted with either a Val, Asp or Cys residue. From an immunologic perspective, these "neo-determinants" may now represent unique and highly specific epitopes for T cell (CD4+ and/or CD8+) recognition in cancer immunotherapy. Evaluation of point-mutated ras as a T-cell epitope could be determined biologically with short synthetic peptides that precisely mimic those altered sites. Several laboratories have established approaches in both murine and human systems to evaluate the point-mutated ras p21 oncogene product as a potential tumor-specific target and characterization of the resulting cellular immune responses. It has been demonstrated that (1) active immunization of mice with the appropriate mutant protein or peptides leads to the production of cytotoxic CD4+ (Th1 subtype) or CD8+ T lymphocytes, which mediate MHC-restricted, antigen-specific lysis of tumor cells in vitro bearing endogenous mutant ras epitopes; and (2) in vitro stimulation of human lymphocytes from some normal individuals or carcinoma patients with mutant ras peptides results in the expansion of CD4+ and CD8+ precursors, which may exhibit cytotoxicity against autologous or MHC-matched, antigen-bearing target cells. Taken collectively, these preclinical findings provide the rationale for the development of potential immunotherapies directed against point-mutated ras oncogene products.

Animals↗
PubMed1995

Admixture of a recombinant vaccinia virus containing the gene for the costimulatory molecule B7 and a recombinant vaccinia virus containing a tumor-associated antigen gene results in enhanced specific T-cell responses and antitumor immunity.

At least two signals are required for the activation of naive T cells by antigen-bearing target cells: an antigen-specific signal, delivered through the T-cell receptor, and a costimulatory signal delivered through the T-cell surface molecule CD28 by its natural ligand B7-1. The immunological benefit of coexpression of B7 with target antigen has been demonstrated with the use of several retroviral systems to transfect antigen-bearing cells. Although engineering recombinant constructs with genes for two or more antigens can mediate the dual expression of those antigens, disadvantages of this approach include the time for construction of each desirable combination and the inability to control differential expression levels of each gene product. An alternative approach would utilize separate constructs that could be admixed appropriately before administration. In this report we describe the functional consequences of the admixture of recombinant vaccinia murine B7-1 (rV-B7) to recombinant vaccinia expressing the human carcinoembryonic antigen gene (rV-CEA). Coinfection of cells resulted in high levels of cell surface expression of both the CEA and B7 molecules. Immunization of mice with various ratios (1:3, 1:1, 3:1) of rV-CEA and rV-B7 demonstrated that an admixture of rV-CEA and rV-B7 at a 3:1 ratio resulted in the generation of optimal CEA-specific T-cell responses. Next, we examined the efficacy of this admixture on antitumor activity. Typically, injection of murine carcinoma cells expressing CEA leads to the death of the host. One immunization of C57BL/6 mice with rV-CEA:rV-B7 (3:1) resulted in no tumor establishment. In contrast, administration of rV-CEA or rV-B7 alone had little or no antitumor effects. These studies demonstrate the advantages of the use of recombinant vaccinia viruses to deliver B7 molecules in combination with a tumor-associated antigen. The availability of the rV-B7 single construct and the ability to alter the B7 ratio could also have potential utility when coinfecting rV-B7 with recombinant vaccinia viruses containing genes for infectious agents or other tumor-associated antigen genes.

Animals↗
PubMed1995

Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21.

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4+ T helper type 1 (Th1) subset. BALB/c mice (H-2d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An alpha beta T cell receptor-positive, CD3+, CD4+, CD8- T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-gamma, tumor necrosis factor and granulocyte-macrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Iad-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4(+)-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II- P815 targets, inhibition of A20 lysis with anti-Iad monoclonal antibodies, and induction of lysis against L cell targets transfected with E alpha A beta d. Independent isolation of a second CD4+ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4+ Th1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.

Animals↗
PubMed1993

Detachment and lysis of adherent target cells by CD4+ T cell clones involve multiple effector mechanisms.

Detachment of adherent targets from their substratum is a unique activity of both CD8+ and CD4+ T cells. Recently, we have demonstrated that with mixed, alloreactive populations, CD4+ T-cell-mediated detachment is kinetically distinct from lysis and requires protein synthesis. Heterogeneity of effector phenotypes precluded further elucidation of the mechanism of detachment at the mixed population level. Here, we examine further the mechanism of target cell detachment by CD4+ cells using clones which differ in lytic efficiency and demonstrate that detachment of adherent targets from their substratum (1) may result from either protein synthesis-dependent or independent pathways, which can be correlated with differences in clonal lytic phenotype, (2) is contact dependent and does not involve soluble factors, (3) can be pharmacologically uncoupled from IL4 production, as a measure of cytokine release, and (4) fails to correlate with perforin expression by immunocytochemistry. Finally, isolated granule preparations are unable to mediate detachment independent from lysis.

Animals↗
PubMed1993

Separation of CD4+ functional responses by peptide dose in Th1 and Th2 subsets expressing the same transgenic antigen receptor.

CD4+ T cells from alpha beta-TCR transgenic mice were used to explore the effect of antigen dose on various functional activities. This transgenic system provides for a source of antigen-specific T cells with the same TCR which can develop into Th1 and Th2 phenotypes under controlled conditions. Of particular interest was to determine if detachment of adherent targets from their substrate could be separated from other functional activities by either antigen dose or functional subset. Using this model, we have found that (a) Th1 and Th2 phenotypes exhibit a similar peptide dose-dependent pattern of detachment and lysis, although Th2 are generally less lytic on both fibroblast and lymphoma targets; (b) detachment and lysis can be dissociated from cytokine production by low peptide doses; (c) detachment and lysis can be physiologically as well as temporally and pharmacologically uncoupled as most targets recovered after detachment by Th2 effectors retain their capacity to proliferate.

Animals↗
PubMed1991

CD4+ T lymphocyte-induced target cell detachment. A model for T cell-mediated lytic and nonlytic inflammatory processes.

We have been exploring the hypothesis that T lymphocytes have the potential to mediate immune damage through nonlytic disruption of tissue organization. In this report, we have examined the ability of purified, primary cultures of alloreactive CD4+ T cells to mediate Ag-specific target cell detachment and/or lysis of L cell lines transfected with MHC class II determinants. Using this model, we demonstrate that: 1) MHC class II-specific CD4+ T cells can cause detachment as a distinct event of the E:T interaction, although the pathways or mechanisms involved appear to be different from those utilized by MHC class I-specific CD8+ T cells; 2) detachment and lysis by CD4+ T cells are distinct activities that involve different functional requirements: 3) CD4+ T cell-induced detachment is initiated by direct cell-cell interaction, independent of TNF-alpha/beta; 4) CD4+ T cell-mediated lysis can be accomplished by TNF-alpha/beta-dependent and independent pathways; and 5) the nature of a particular target cell response to alloreactive CD4+ T cell attack reflects its intrinsic susceptibility to one or more potential effector mechanisms.

Animals↗
PubMed1989

Cytotoxic T lymphocyte-induced loss of target cell adhesion and lysis involve common and separate signaling pathways.

Recently, we demonstrated that an early event in the CTL-target cell (TC) interaction is loss of TC adherence to substrate. This loss of adhesion is Ag-specific, but distinct from the lytic event because it can ensue in nominally Ca2+-free medium. In this study, we examine further the mechanism of CTL-induced loss of adhesion, concentrating mainly on the signal transduction pathway. Based on the differential sensitivity of CTL to extracellular Ca2+, protein kinase C activation/depletion and inhibition by anti-Lyt-2 (CD8) or anti-CTL receptor (TCR) reagents, we demonstrate that CTL-induced loss of adhesion can be initiated through multiple activation pathways. Although CTL-mediated lysis is restricted to a Ca2+ and protein kinase C-dependent signaling mechanism, CTL-induced loss of adhesion is initiated in the presence or absence of extracellular Ca2+ or functional protein kinase C activity. Furthermore, although under physiologic conditions, anti-CD8 or anti-TCR reagents strongly block both CTL activities, under non-lytic conditions, they fail to inhibit the ability of CTL to promote loss of adhesion. These findings implicate the participation of additional CTL-TC ligand interactions resulting in loss of adhesion, and thus, provide further evidence to support the hypothesis that CTL-induced loss of adhesion can be initiated through multiple triggering pathways.

Animals↗
PubMed1988

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities.

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1986

The functional loss of human natural killer cell activity induced by K562 is reversible via an interleukin-2-dependent mechanism.

In a recent study, we evaluated the functional status of human natural killer (NK) cells after their interaction with the NK-sensitive tumor target cell (TC), K562. We demonstrated that effector cells (EC), after treatment with K562 for 4 hr, lost greater than 90% of their original lytic activity. In this investigation, we examined whether this functional loss of NK cell activity represented an irreversible event in the NK lytic mechanism. Initial studies focused on the ability of K562-inactivated EC (ECi), which had been separated from their TC, to recover cytolytic activity following an 18-hr incubation. Our results indicated that ECi recovered 28% of their lytic activity in complete medium (CM) alone, 64% in CM containing interferon-beta (IFN-beta), and 91% in CM supplemented with interleukin 2 (IL-2). Analysis of the data revealed, however, that neither IFN-beta nor IL-2 simply boosted the lytic capacity of NK cells which initially escaped inactivation, but also, each cytokine affected the lytic capabilities of EC that were either truly inactivated by K562 or precursor NK (pre-NK) cells. Thus, to evaluate further the basis of IFN-beta and IL-2-induced ECi augmentation, we first treated the EC with IFN-beta or IL-2 prior to their interaction with K562 so that pre-NK cell subsets would be promoted to fully competent NK cells. Both pretreated EC preparations, after interacting with K562 for 4 hr, lost greater than 90% of their original lytic activities. NK inactivation did not result from cell death nor reflect alterations in conjugate formation or the percentages of Leu-7- and Leu-11-positive EC. IL-2-pretreated ECi, as did ECi, regained some lytic activity after incubation in CM alone, but recovered significantly more activity in CM containing IFN-beta or IL-2. In contrast to the restimulation profiles obtained for ECi and IL-2-pretreated ECi, IFN-pretreated ECi regained lytic function after incubation with IL-2, but not appreciably with IFN-beta or in CM alone. Overall, these findings suggest that EC, either untreated or pretreated with IFN-beta or IL-2, significantly lose their lytic capabilities following interaction with K562 while retaining their ability to bind to the TC; IFN-beta acts predominantly on pre-NK cells, but not on ECi; and IL-2 appears to play an important role in restoring lytic potential to functionally inactive NK cells.

Cells, Cultured↗
PubMed1985

Evidence for an early calcium-independent event in the activation of the human natural killer cell cytolytic mechanism.

In this study, we examined the functional status of human natural killer (NK) cells after their direct interaction with the NK-sensitive tumor target cell (TC), K562. Human peripheral blood lymphocytes depleted of adherent cells were incubated for 4 hr with unlabeled K562 cells at an effector cell (EC) to TC ratio of 2:1. After incubation, the EC were separated from the TC via centrifugation over a single-step Percoll gradient. K562-treated and separated EC were subsequently shown to be unable to lyse fresh K562 TC when retested in the standard chromium-release assay. Kinetic studies revealed that greater than 90% inactivation of NK cell-mediated cytotoxicity (CMC) could be achieved within 2 hr. Inactivation of NK-CMC by K562 was not caused by a specific loss of NK cells, as detected by changes in the expression of two NK cell-associated markers, Leu-7 and Leu-11, or to alterations in EC viability and target binding cell capacity. Interestingly, NK inactivation also occurred in medium devoid of extracellular calcium, although parallel testing of NK-CMC in the same medium resulted in no chromium release. NK inactivation, however, was significantly prevented when the EC and TC were co-incubated at 4 degrees C, or in medium without magnesium. Additional studies revealed that inactivation of NK-CMC could be achieved with another NK-sensitive, but not with an NK-resistant TC. Overall, we demonstrated that NK cells rapidly lost their lytic potential after direct interaction with a sensitive TC, although the cells remained viable, expressed the same percentage of Leu-7 and Leu-11, and could still bind the TC; and NK inactivation occurred in the absence of extracellular calcium, but not when EC and TC were incubated in medium without magnesium. These latter results provide evidence for an early event in the activation of human NK cells that is binding dependent, temperature sensitive, and independent of extracellular calcium.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Compared mechanisms of tumor cytolysis by human natural killer cells and activated polymorphonuclear leukocytes.

The mechanism(s) of natural killer (NK) cell-mediated cytotoxicity (CMC) remains largely unknown. In this study, we investigated the possibility of human NK cells to exhibit an oxidative burst (OB) after stimulation by K562, an NK-sensitive target cell (TC). The addition of catalase (CAT) or superoxide dismutase (SOD) to the NK-mediated cytotoxic assay had no effect on NK-CMC. In contrast, CAT and SOD effectively modulated the cytotoxicity mediated by phorbol-12-myristate-13-acetate (PMA)-activated polymorphonuclear leukocytes (PMNL) against three different tumor TC, including K562. CAT abrogated, while SOD enhanced PMA-activated PMNL-mediated cytotoxicity. The synergistic effect of SOD and PMA was suppressed in a dose-dependent fashion by CAT. Furthermore, by chemiluminescence (CL) and SOD-inhibitable reduction of cytochrome c, we failed to detect an OB associated with K562-stimulated NK cells. PMNL, however, rapidly responded to PMA (10 ng/ml), generating almost 10(6) cpm within 20 min and 26.7 nM O-2/10(6) cells/30 min, as detected by CL and reduction of cytochrome c, respectively. Finally, K562 alone, at cell concentrations corresponding to effector cell:target cell (EC:TC) ratios of 1:1 and 1:10, reduced cytochrome c, but this reduction was not inhibited by SOD, thus suggesting a diaphorase activity. Overall, we show that: a) tumor cell destruction by human NK cells and by PMA-activated PMNL is mediated by different mechanisms; and b) NK-CMC against a sensitive TC does not involve an OB.

Catalase↗
PubMed1983

Mechanism of action of phorbol myristate acetate on human natural killer cell activity.

We have investigated the kinetics of inhibition and regeneration of human natural killer (NK) cell-mediated lysis of K562, a human erythroleukemia cell line, by the potent tumor-promoting agent phorbol-12-myristate-13-acetate (PMA). It is shown that PMA inhibits NK cell-mediated cytotoxicity (CMC) in a dose-dependent manner whether the compound is present throughout the 4-hr cytotoxic assay or the effector cells (EC) are pretreated with PMA. Pretreatment of the target cells (TC) with PMA produced a different profile of NK activity suggesting that PMA inhibition of NK-CMC is primarily due to the inactivation of EC. PMA-induced inhibition of NK-CMC does not affect TC binding and is not circumvented by compounds that enhance intracellular levels of cyclic guanosine monophosphate (cGMP) or calcium. Furthermore, and contrary to a recent report, PMA-induced inhibition of NK-CMC is independent of monocytes. Finally, kinetic studies revealed that PMA-induced inhibition of NK-CMC occurs rapidly and is fully reversible provided that "regenerated EC" are thoroughly washed, prior to the cytotoxic assay, to rid the cell suspension of residual PMA. The potential implications of these results to the currently accepted theory of TC destruction by NK cells, the stimulus-secretion model, are discussed.

Calcimycin↗
PubMedDate not supplied

Mechanism of K562-induced human natural killer cell inactivation using highly enriched effector cells isolated via a new single-step sheep erythrocyte rosette assay.

In this study, we used preparations highly enriched in human natural killer (NK) cells to further characterize the mechanism of target-cell-induced NK inactivation. Highly enriched populations of NK cells were obtained by a newly developed, single-step sheep red blood cell rosette assay. This method, which did not require any incubation steps to facilitate cell contact, permitted a rapid and efficient isolation of NK cells from adherent-cell-depleted peripheral blood lymphocytes. The non-rosetted cells had high NK activity, possessed large granular lymphocyte (LGL) morphology and expressed the NK-associated antigens Leu-11a, Leu-7, OKM1 and NKH-1. In contrast, the rosetted cells had significantly lower NK activity, possessed typical lymphocyte morphology and expressed the T-cell-associated marker OKT3. Next, we examined the ability of these NK-enriched effector cells (ECc) to become inactivated by K562. Functional studies revealed that ECc lost greater than or equal to 95% of their lytic capacity following incubation with K562 at a ratio of 2/1 for 6 h. However, to achieve this level of inactivation, it was essential that the cell suspension be gently mixed every 90-120 min. Inactivation was not due to cell death and did not reflect changes in the percentages of cells bearing the Leu-11a, Leu-7, OKM1 and NKH-1 antigens, but was associated with an increase in cell surface concentration of OKM1. As judged by gross morphology, the percentages of LGL in ECc before and after treatment with K562 were essentially the same. Finally, K562-treated ECc also lost their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), suggesting that both NK-cell-mediated cytotoxicity and ADCC may involve a common lytic pathway.

Animals↗