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V H Engelhard

Publications and source records attributed to V H Engelhard.

At least 109 records · Page 6Linked to original sources
PubMed1988

Early events in target-cell lysis by cytotoxic T cells.

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

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PubMed1987

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity.

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.

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PubMed1987

The role of K+ in the regulation of the increase in intracellular Ca2+ mediated by the T lymphocyte antigen receptor.

The regulation of the increase in intracellular calcium ([Ca2+]i) occurring in cytolytic T lymphocytes (CTLs) upon their interaction with antigen was examined. This [Ca2+]i increase and lytic function were insensitive to verapamil, a Ca channel blocker. An antigen-independent increase in [Ca2+]i was not induced by depolarization of CTLs with excess extracellular K+, suggesting that Ca2+ influx is not mediated by the ubiquitous voltage-gated Ca channel. The antigen-induced [Ca2+]i increase was inhibited by prior membrane hyperpolarization with valinomycin. Hyperpolarization occurred under normal circumstances in CTLs exposed to antigen-receptor-specific antibodies. This potential change was Ca2+-dependent and inhibited by K channel blockade. Conversely, K channel blockade augmented the antigen-specific [Ca2+]i increase while markedly decreasing the K+ efflux associated with CTL lytic function. Therefore, either membrane potential or intracellular K+ regulates the antigen-specific [Ca2+]i increase in CTLs.

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PubMed1987

Demonstration of a calcium influx in cytolytic T lymphocytes in response to target cell binding.

By using the Ca2+-sensitive dye indo-1, an antigen-specific increase in intracellular Ca2+ in cloned cytolytic T lymphocytes (CTL) was measured under conditions that were permissive for T cell-mediated cytolysis. To synchronize lethal hit delivery in a suspension of effector and target cells, a modification of the cation pulse method in which Ca2+ is added to preformed conjugates of CTL and target cells was used. Conjugate formation was unaffected by the absence of extracellular Ca2+ under these conditions. Lytic activity of these cloned CTL was markedly reduced in the absence of extracellular Ca2+ and was restored upon Ca2+ repletion. When indo-1-loaded CTL were preincubated with target cells in the absence of extracellular Ca2+, a marked antigen-specific increase in indo-1 fluorescence, indicative of an increase in intracellular Ca2+, was observed after repletion of extracellular Ca2+. This increase in intracellular Ca2+ was shown to be due solely to changes in the CTL and not the target cell within the time course of the experiment, and results from the influx of extracellular Ca2+. Antibody to the T cell receptor for antigen also evokes a similar increase in intracellular Ca2+ in CTL under these conditions. This method provides a means for the direct examination of the response of CTL to cellular antigen as well as soluble antibody and is a versatile and valuable tool for the study of CTL function.

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PubMed1987

Bone marrow irradiation chimeras in the BB rat: evidence suggesting two defects leading to diabetes and lymphopoenia.

A series of bone marrow irradiation chimeras were constructed in an attempt to determine the site of the defect(s) leading to diabetes and/or lymphopoenia in the BB rat. In BB rats that were lethally irradiated and reconstituted with T-cell-depleted Wistar-Furth (WF) rat bone marrow, the incidence of diabetes was reduced, and in animals treated with WF bone marrow at less than 44 days of age, the disease was completely prevented. Such animals demonstrated normal lymphocyte counts in peripheral blood, and normal lymphocyte function (as indicated by mixed lymphocyte response), but retained an abnormal T-cell subset distribution only partially improved above that of diabetes-prone BB rats. The incidence of diabetes in these irradiated chimeras was significantly reduced compared to the incidence in BB rats irradiated at the same age but reconstituted with bone marrow from BB rats. In WF rats that were lethally irradiated and reconstituted with T-cell-depleted bone marrow from overtly diabetic BB rats, no diabetes was induced. Such animals demonstrated normal lymphocyte counts in peripheral blood, normal lymphocyte function, and normal T-cell subset distributions. Overall, these results suggest two defects leading to diabetes and/or lymphopoenia in the BB rat. One of these occurs at the level of the bone marrow stem cell while the other resides in the T-cell differentiative environment.

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PubMed1986

Derivation of cytotoxic T cell lines that express T cell receptor but exhibit reversible loss of stable antigen binding.

Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations.

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PubMed1986

HLA antigens expressed on murine cells are preferentially recognized by murine cytotoxic T cells in the context of the H-2 major histocompatibility complex.

The frequency of murine cytotoxic T lymphocytes (CTL) capable of responding to HLA antigens expressed on human or murine cells was determined by limiting dilution analysis. HLA antigens expressed on human cells stimulated CTL with a precursor frequency of about 1 in 2 X 10(5) spleen cells in primed mice, over two orders of magnitude weaker than a primary allogeneic response. There was a 10-fold increase in the frequency of precursors responding to HLA antigens when they were expressed on murine cells. It was determined that the increased frequency of responders was due to CTL that could only recognize HLA antigens on the syngeneic murine line to which they had been stimulated and that these CTL could not lyse any other HLA expressing murine cells of different H-2 haplotypes. The lytic activity of these CTL was inhibited by H-2b-specific antibodies. These results indicate that such CTL recognize HLA antigens in the context of the H-2 major histocompatibility complex. The magnitude and specificity of CTL responses to xenoantigens are discussed in the context of a model for T-cell interactions with major histocompatibility antigens.

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PubMed1986

Prevention of diabetes in BB rats. I. Evidence suggesting a requirement for mature T cells in bone marrow inoculum of neonatally injected rats.

Injection of major histocompatibility complex (MHC)-compatible bone marrow cells from normal animals into neonatal BB rats resulted in a striking decrease in incidence of diabetes and restoration of concanavalin A (ConA) and mixed lymphocyte responses. However, injection of bone marrow cells pretreated with anti-rat thymocyte antiserum plus complement to remove mature T cells had no effect on incidence of disease, suggesting that mature T cells in the bone marrow inoculum were responsible for prevention of diabetes. Because the decreased incidence of diabetes in rats injected with untreated bone marrow appeared to be unrelated to the extent of lymphopenia in these animals, the involvement of T cells in the onset of diabetes must reflect a defect in the normal function of these cells rather than their absolute number. Approximately 50% of the W3/13+ cells in the spleens of BB rats lacked the OX-8 and W3/25 T cell subset markers. The identity of this W3/13+, OX-8-, W3/25- blank subset remains to be established. Our results, interpreted in light of studies from the other laboratories, suggest the existence of multiple abnormalities in the BB rat, including the presence of T cells as effector or helper cells that augment onset of disease and the absence of a regulatory T cell circuit that could prevent the disease.

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PubMed1986

Reorientation and fusion of cytotoxic T lymphocyte granules after interaction with target cells as determined by high resolution cinemicrography.

The interaction of murine cytotoxic T lymphocyte (CTL) clones with human lymphoblastoid target cells was studied in thin preparations by using high resolution cinemicrography. CTL not bound to target cells were morphologically polar, possessing a broad leading edge containing the nucleus, and a tapered tail containing a large number of granules. The CTL were observed to move by the extension of pseudopods from the leading edge. Initial contact with a target cell was made via the leading edge of the CTL. If the human target cell expressed the appropriate HLA antigen, distinct morphologic changes occurred in the CTL as early as 2 min after initial contact. The CTL rounded up, and the nucleus moved from a position adjacent to the zone of contact to be replaced by the cytoplasmic granules. Redistribution of the granules was completed as early as 10 min after initial contact was made. These morphologic changes did not occur when the CTL made contact with other CTL, or with target cells that did not express the appropriate HLA antigens. In studies that make use of Nomarski optics, an apparent fusion of CTL cytoplasmic granules with the membrane in the vicinity of the target cell contact area was observed 4 min after binding, and before granule reorientation was complete. These data provide direct evidence for the occurrence of both reorientation of the cytoplasmic contents and granule fusion in CTL with a time course similar to that of administration of the lethal hit.

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PubMed1985

Multiple epitopes on human and murine cells expressing HLA-B7 as defined by specific murine cytotoxic T cell clones.

Eleven cytotoxic T lymphocyte (CTL) clones were derived from C57BL/6 spleen cells immunized with HLA-B7 expressing human lymphoblastoid cell lines. Reactivity against HLA-B7 was initially established because the clones lysed 2 target cells that shared only HLA-B7 with the immunizing cell line and they did not lyse five other cell lines that were HLA-B7 negative but expressed other class I or class II antigens found on the immunizing cell. Six of the clones were subsequently shown to lyse all tested HLA-B7-positive B and T lymphoid cell lines, peripheral blood lymphocytes, and a murine L cell that expressed HLA-B7 as a consequence of DNA-mediated gene transfer. On the basis of the inability of the clones to lyse a panel of HLA-B7-negative cell lines, up to 18 other class I antigens could be eliminated as being cross-reactively recognized. However, two of the clones recognized a single HLA-B7-negative cell line. It is suggested that in these cases the clones were cross-reactively recognizing the HLA-B27 or HLA-B40 antigens that were present on these target cells. The remaining five CTL clones failed to lyse one out of seven tested HLA-B7-positive lymphoid lines (either RPMI-1788 or DR1B) and failed to lyse peripheral blood lymphocytes from one out of three tested HLA-B7-positive individuals. These five clones also did not recognize the HLA-B7-positive murine L cell. However, based on analysis with a large target cell panel, the reactivity pattern of these five clones could only be correlated with recognition of HLA-B7. This conclusion is further supported by antibody-blocking studies to be reported elsewhere. As before, lysis of single HLA-B7-negative target cells by two of the clones could be ascribed to recognition of HLA-B27 or HLA-B40. The results show that murine clones raised against HLA-B7 exhibit a high degree of specificity for determinants that are unique or largely confined to the HLA-B7 alloantigen. In addition, these clones define different antigenic determinants on the molecule. Thus, such clones appear to be excellent candidates for use as human tissue typing reagent. The results further show that there is a strong correlation between recognition of particular HLA-B7-positive human cell lines and recognition of the HLA-B7 expressing murine L cell. Possible reasons for such a correlation and their relationship to the general phenomenon of CTL recognition are discussed.

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PubMed1985

Construction of novel class I histocompatibility antigens by interspecies exon shuffling.

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.

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PubMed1984

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

Animals↗
PubMed1984

Introduction of HLA-A/B antigens into lymphoid cell membranes by cell-liposome fusion.

Conditions were optimized for surface binding by mouse lymphblastoid cell lines of liposomes containing purified human histocompatibility antigens. Antigens were reconstituted into lipid vesicles by detergent dialysis, and were incubated with the acceptor cell lines. After washing, the amount of HLA antigen on the cell surface was quantitated by double antibody radioimmunoassay. Uptake and surface expression was shown to be dependent upon vesicle phospholipid composition, vesicle:cell ratio, acceptor cell line, and stage of cell growth. These studies indicate that by using vesicles composed of 50% phosphatidylethanolamine/50% phosphatidylserine it is possible to introduce into the mouse EL4 cell surface about 70% of the amount of HLA expressed normally on the human lymphoblastoid line JY. The antigen is stably expressed on the surface for several hours and appears to be integrated into the cell membrane, as assessed by both fluorescence microscopy and susceptibility to lysis by using anti-HLA antibody and complement. The method used has the advantage over previously described procedures of not requiring the use of fusogenic proteins or agents such as polyethylene glycol or lysophospholipids, which might perturb overall membrane structure or properties.

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PubMed1983

Xenogeneic cytotoxic T-cell clones recognize alloantigenic determinants on HLA-A2.

Long-term murine cytotoxic T-cell clones arising in response to stimulation with human lymphoblastoid cells and reactive with the HLA-A2 antigen are characterized. These clones distinguish between HLA-A2 and 21 other serologically defined HLA-A and -B antigens. In addition, most clones discriminate between prototypical HLA-A2 antigens, expressed by the majority of HLA-A2-positive individuals, and variant HLA-A2 antigens, which are serologically identical with the prototype, but distinguishable by human cytotoxic T cells and by biochemical analysis. This discrimination is reflected as an inability to cause any significant lysis of variant HLA-A2-expressing target cells at effector-to-target ratios 10- to 100-fold greater than those giving 50% lysis of prototype HLA-A2-expressing cells. By screening a panel of serologically HLA-A2-positive cells, a new variant HLA-A2-expressing cell line has been defined. The recognition patterns of these xenogeneic clones are suggested to reflect recognition of alloantigenic polymorphic determinants. Based on the strong bias in the xenogeneic T-cell repertoire for such determinants, we propose a model for T-cell recognition of class I products of the major histocompatibility complex.

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PubMed1983

Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.

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PubMed1980

Specificity of mouse cytotoxic T lymphocytes stimulated with either HLA-A and -B or HLA-DR antigens reconstituted into phospholipid vesicles.

Distinct populations of murine cytolytic T lymphocytes (CTL) were elicited from primed spleen cells by preparations of HLA-A and -B (HLA-A,B) or HLA-DR antigens reconstituted into phospholipid vesicles. These populations could be distinguished by both antiserum blocking and by patterns of cytolysis of a panel of target cells. Cytolysis by CTL stimulated with liposomes that contained HLA-A2 and HLA-B7 antigens could only be blocked by antiserum against HLA-A,B antigens but not by antiserum against HLA-DR antigens. The inverse pattern was seen with HLA-DR-stimulated CTL. When compared with a panel of target cells expressing various HLA-A,B or HLA-DR allospecificities, the strongest CTL reactivity was seen toward those cells that bore the same allospecificities as those presented on the liposomes. Target cells that expressed cross-reactive specificities and unrelated specificities were recognized much less well. The implications of the results for the mechanism of CTL stimulation by liposomes, as well as the relationship between allogeneic and xenogeneic CTL recognition, are discussed.

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