Biomedical subjects

M F Mescher

Publications and source records attributed to M F Mescher.

At least 19 recordsLinked to original sources

PubMed2001

Signaling alterations in activation-induced nonresponsive CD8 T cells.

Costimulation-dependent production and autocrine use of IL-2 by activated CD8 T cells results in initial clonal expansion, but this is transient. The cells quickly become anergic, unable to produce IL-2 in response to Ag and costimulation, irrespective of the form of costimulation. This activation-induced non-responsiveness (AINR) differs from "classical" anergy in that it results despite the cells receiving both signal 1 and signal 2. AINR cells can still proliferate in response to exogenous IL-2, but can no longer produce it. Other TCR-mediated events including cytolytic function and IFN-gamma production are not affected in the AINR state. To characterize the mechanism(s) responsible for lack of IL-2 production in CD8 T cells in the AINR state, microspheres bearing immobilized anti-TCR Abs or peptide-MHC complexes, B7-1, and ICAM-1 were used to provide well-defined stimuli to the cells. Comparison of normal and AINR cells revealed that in AINR cells extracellular signal-regulated kinase (ERK) is upregulated more transiently, Janus kinase activation is substantially reduced, and activation of p38 is eliminated. PMA and ionomycin restored proliferation and IL-2 production in AINR cells, indicating a signaling defect upstream of Ras and protein kinase C. Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2 mRNA expression and proliferation of both peptide-MHC/B7-1/ICAM-1-stimulated normal cells and PMA/ionomycin-stimulated AINR cells. Together these results demonstrate that activation of at least ERK and p38 is essential for IL-2 production by CD8 T cells and that up-regulation of these mitogen-activated protein kinases, along with Janus kinase, is defective in AINR cells.

Animals↗

PubMed2001

LFA-1-mediated costimulation of CD8+ T cell proliferation requires phosphatidylinositol 3-kinase activity.

LFA-1 binding to ICAM-I provides a costimulatory signal for CD8(+) T cell activation that results in increased IL-2 mRNA levels and protein production to support proliferation. CD28 binding to its B7 ligands has the same effect, and the two costimulatory receptors activate some of the same intracellular signaling events, including up-regulation of phosphatidylinositol (PI) 3-kinase activity. However, costimulation by LFA-1 depends upon the activity of this enzyme, whereas costimulation by CD28 does not, as evidenced by differential effects of specific inhibitors of PI 3-kinase. When cells are costimulated with ICAM-1 in the presence of the inhibitors wortmannin or LY294002, proliferation is blocked, but increases in IL-2 mRNA levels and protein production are not. Costimulation also results in increased surface expression of CD25, which is essential for formation of an active IL-2R. This is blocked by the PI 3-kinase inhibitors when costimulation is via LFA-1 but not when it is via CD28. Finally, IL-2-driven proliferation is not blocked by the inhibitors once CD25 surface expression has increased. Thus, the PI 3-kinase-dependent step in CD8 T cell costimulation by LFA-1 is up-regulation of IL-2R expression. In contrast, CD28 engagement also increases IL-2R surface expression, but the up-regulation does not depend upon PI 3-kinase activity.

Androstadienes↗

PubMed2001

Il-12 enhances CD8 T cell homeostatic expansion.

The size of the T lymphocyte pool is maintained by regulation of T cell production, proliferation, and survival. Under the pressure of a T lymphopenic environment, mature naive T cells begin to proliferate in the absence of Ag, a process called homeostatic expansion. Homeostatic expansion involves TCR recognition of self peptide/MHC ligands, but less is known about the soluble factors that regulate this process. Here we show that IL-12 dramatically enhanced the homeostatic proliferation of CD8 T cells. In contrast, IL-2 had no beneficial effect on homeostatic expansion and, in fact, inhibited T cell expansion induced by IL-12. Using gene-targeted mice, we showed that IL-12 acted directly on the T cells to enhance homeostatic expansion, but that IL-12 cannot override the requirement for TCR interaction with self peptide/MHC ligands in homeostatic expansion. These data indicate that inflammatory cytokines may modulate T cell homeostasis after lymphopenia and have implications for regulation of the T cell repertoire and autoimmunity.

Adjuvants, Immunologic↗

PubMed2001

Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins.

T cell activation results from the engagement of multiple receptors on T cells by their respective ligands on antigen presenting cells. Studies using artificial cell surface constructs have demonstrated that effective T cell response requires that antigen be presented on a solid surface with dimensions that approximate those of an intact cell. In this report, we describe the cloning and expression of recombinant B7-1-Fc and B7-2-Fc proteins and their incorporation onto 5-microm latex microspheres along with renatured peptide-MHC. These microspheres provide a simple and effective method for the in vitro or in vivo stimulation of antigen-specific T cells under precisely controlled antigen and costimulation conditions.

Animals↗

PubMed2001

Role of phosphoinositide 3-kinase in TCR-signaled regulation of CD8-mediated adhesion to class I MHC protein.

CD8 must be activated by signaling through the TCR in order to mediate CTL adhesion. Up-regulation of adhesion to class I protein is shown to be blocked by specific inhibitors of phosphoinositide 3-OH kinase (PI3-K), indicating a critical role for this enzyme in signaling for activation of CD8. A minimal TCR stimulus that activates CD8 does not result in a detectable increase in total cellular PI3-K activity, but an increase in PI3-K activity associated with p59(fyn) kinase can be detected. Genistein blocks this increase concomitantly with blocking the activation of adhesion, suggesting that activation of fyn-associated PI3-K is downstream of TCR-dependent activation of protein tyrosine kinase(s) in the signaling pathway that leads to up-regulation of CD8-dependent adhesion. Treatment of cells with phorbol ester also blocks the TCR-dependent increase in fyn-associated PI3-K and inhibits CD8-dependent adhesion. This suggests a feedback model for deactivation of CD8 adhesion to allow target cell release by CTL and recycling to kill additional targets. In contrast, phorbol ester treatment up-regulates integrin-mediated adhesions, suggesting complex cross-talk between the TCR and the different adhesion/cosignaling receptors during the binding and killing of antigen-bearing targets.

Animals↗

PubMed1999

Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo.

CD8+ T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the 1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B2+ cells in the draining lymph nodes (DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in a large clonal expansion of 1B2+ cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B2+ cell numbers decreased in the DLN and increased in the spleen and blood. 1B2+ cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B2+ T cell activation and clonal expansion. Thus, CFA or IL-12 can enhance Ag-specific CD8+ T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can support activation and prevent tolerance induction.

Adjuvants, Immunologic↗

PubMed1999

CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation.

CD8+ T cells stimulated in vitro with anti-TCR mAb and B7-1 or ICAM-1 produce IL-2 and clonally expand. Effector function is acquired within 3 days, but proliferation ceases and the cells begin to die by apoptosis. Stimulation in vivo with B7-1-expressing allogeneic tumor results in the same sequence of events with a comparable time course. In both cases, the cells become anergic within 3 or 4 days of responding; they can no longer respond by producing IL-2 and proliferating, but can still be stimulated to proliferate in response to exogenous IL-2. This activation-induced nonresponsiveness (AINR) is not simply a consequence of ongoing cell death; cytokines that promote survival (IL-7 or IFN-alpha) or proliferation (human IL-2) do not restore the ability to produce IL-2 in response to costimulation. Although similar to the anergy described for CD4+ T cell clones, AINR differs in that it results from an initial stimulation with both signal 1 and signal 2. AINR appears to be an aspect of the normal differentiation of fully stimulated CD8+ T cells. It is probably important in regulating CTL responses; it limits the initial T helper-independent response and converts it to a response that requires T cell help to be sustained and further expanded. When the initial helper-independent response is not sufficient to clear Ag, and if help is not available, AINR likely results in tolerance to the Ag.

Abatacept↗

PubMed1999

Signaling pathways activated by leukocyte function-associated Ag-1-dependent costimulation.

LFA-1 binding to ICAM-1 can enhance TCR-dependent proliferation of T cells, but it has been difficult to distinguish contributions from increased adhesion, and thus TCR occupancy, versus costimulatory signaling. Whether LFA-1 ligation results in generation of a unique costimulatory signal(s) distinct from those activated by the TCR has been unclear. Using purified ligands, it is shown that ICAM-1 and B7. 1 provide comparable costimulation for proliferation of CD8+ T cells, and that both ligands up-regulate the activities of phosphatidylinositol 3-kinase, sphingomyelinase, and c-Jun NH2-terminal kinase (JNK). These pathways are distinct from those activated by the TCR, and have previously been implicated in up-regulating IL-2 production in response to CD28-B7 interaction. Thus, under conditions in which ICAM-1 provides costimulation of proliferation, LFA-1 ligation activates some of the same signaling pathways as does CD28 ligation. LFA-1 and CD28 do not act identically, however, as indicated by differential sensitivity to inhibitors of phosphatidylinositol 3-kinase; LFA-1-dependent costimulation of proliferation is inhibited, while CD28-dependent costimulation is not. Given the broad distribution of class I and ICAMs on many cell types, the ability of LFA-1 to provide costimulatory signals has implications for where and how CD8+CTL may become activated in response to an antigenic challenge.

Androstadienes↗

PubMed1999

Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells.

The effects of inflammatory cytokines on naive T cells have been studied using MHC protein/peptide complexes on microspheres, thus avoiding the use of APCs whose functions may be affected by the cytokines. IL-1, but not IL-12, increased proliferation of CD4+ T cells in response to Ag and IL-2, which is consistent with effects on in vivo priming of CD4+ cells. In contrast, proliferation of CD8+ T cells to Ag and IL-2 required IL-12, and IL-12 replaced adjuvant in stimulating an in vivo response to peptide. These results support a model in which distinct inflammatory cytokines act directly on naive CD4+ and CD8+ T cells to provide a third signal, along with Ag and IL-2, to optimally activate differentiation and clonal expansion.

Animals↗

PubMed1999

Control of syngeneic tumor growth by activation of CD8+ T cells: efficacy is limited by migration away from the site and induction of nonresponsiveness.

Activation of Ag-specific CD8+ T cells in response to syngeneic tumor has been visualized by adoptive transfer of CD8+ T cells from OT-I mice, with a transgenic TCR specific for H-2Kb and an OVA peptide, into Thy-1 congenic recipients. Intraperitoneal challenge with E.G7, the EL-4 thymoma transfected with OVA, results in activation and clonal expansion of the OT-I cells in the peritoneal cavity and transient control of tumor growth. However, within 2 days after becoming activated, the OT-I cells migrate out of the peritoneal cavity into the spleen and lymph nodes, and tumor growth resumes in the peritoneal cavity. The OT-I cells in lymph nodes and spleen have lytic effector activity, but exhibit split anergy in that they cannot proliferate in response to Ag unless exogenous IL-2 is provided. The failure to remain at the tumor site and continue to control tumor growth is not due to selection of Ag loss variants or development of suppression. These results suggest that effective CD8-targeted immunotherapy may depend less on enhancing the initial activation and more on sustaining the response at the appropriate location and/or reactivating cells that have left the site of tumor growth and become nonresponsive.

Animals↗

PubMed1999

ICAM-1 and B7-1 provide similar but distinct costimulation for CD8+ T cells, while CD4+ T cells are poorly costimulated by ICAM-1.

The function of purified ICAM-1 in costimulating CD4+ and CD8+ T cell responses has been directly compared to that of B7-1 in a model system that minimizes contributions of other receptor-ligand interactions. While B7-1 costimulates both subsets of T cells, ICAM-1 is much more effective in the costimulation of CD8+ cells. ICAM-1 also synergizes with B7-1 for the induction of IL-2 production in CD8+ but not CD4+ T cells. These differences are not explained by differences in LFA-1 receptor expression on the two subsets of T cells. The CD8+ T cell response to ICAM-1 costimulation is associated with increased proliferation and IL-2 production at levels similar to those seen with B7-1 costimulation, but clonal expansion in response to ICAM-1 is not as great due to decreased cell survival. ICAM-1-mediated costimulation is effective for both naive and memory CT8+ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM-1-dependent, B7-independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.

Animals↗

PubMed1999

CTLA-4 blockade reverses CD8+ T cell tolerance to tumor by a CD4+ T cell- and IL-2-dependent mechanism.

A tumor-specific CD8+ T cell response was studied using adoptive transfer of OT-I TCR transgenic cells. Upon i.p. challenge with E.G7 tumor, OT-I cells undergo CD4+ T cell-independent expansion at the tumor site and develop lytic function. Before tumor elimination, however, they leave the peritoneal cavity (PC) and appear in the LN and spleen where they exhibit "split anergy" and cannot further proliferate to antigen. Administering anti-CTLA-4 mAb early caused sustained OT-1 expansion in the PC, and late administration caused the OT-I cells to return to the PC and further expand; in both cases, tumor was controlled. These effects required CD4+ T cells and IL-2 and appear to result from reversal of the nonresponsive state of the CD8+ T cells.

Abatacept↗

PubMed1998

Qualitative differences between naive and memory T cells make a major contribution to the more rapid and efficient memory CD8+ T cell response.

CD8+ T cells are present at a higher frequency following a primary response, and these memory cells exhibit qualitative differences from naive cells. The importance of these differences vs increased precursor frequency in making a memory response more rapid and efficient has been unclear. Adoptive transfer of 2C TCR transgenic CD8+ T cells into normal recipients, followed by i.p. challenge with allogeneic P815 tumor, results in a long-lived memory population that includes both endogenous host CD8+ T cells and 2C cells. The 2C cells can be identified, using 1B2 mAb specific for the TCR, and thus used as an indicator of the properties of the memory cells. The memory cells have a heterogeneous surface phenotype, and their distribution in lymphoid organs, blood, and peripheral sites is distinct from that of naive cells. Upon rechallenge with Ag, memory cells access the peritoneal cavity much more rapidly than do naive cells (12 h vs 5 days). This appears to result from a requirement for naive cells to interact with Ag before they can efficiently migrate to inflammatory sites, while this is not required for memory cells. In addition, memory cells exhibit some cytolytic activity before rechallenge with Ag, and potent cytolytic activity is present in the peritoneal cavity within 12 h of rechallenge. Comparison of primary and memory responses in mice having similar frequencies of Ag-specific precursors demonstrated that the more rapid migration and the immediate effector function of at least some memory cells contribute very substantially to making a memory response at a peripheral site more rapid and efficient.

Adoptive Transfer↗

PubMed1998

CD8+ memory T cells (CD44high, Ly-6C+) are more sensitive than naive cells to (CD44low, Ly-6C-) to TCR/CD8 signaling in response to antigen.

Memory CD8+ T cells from mice previously primed with alloantigen (alloAg) can respond in vitro to IL-2 and purified class I alloAg presented on microspheres, while no response can be detected using cells from naive mice. Similar results have been obtained using cells from OT-1 mice expressing a transgenic TCR that is specific for OVA(257-264) (SIINFEKL) peptide bound to H-2Kb. A population of resting memory cells (defined on the basis of low forward scatter and CD44high, Ly-6C+, CD25-, CD69-surface phenotype) that is present in the OT-1 mice exhibits a substantially higher sensitivity to Ag-stimulation than do naive cells (CD44low, Ly-6C-) expressing the same TCR. CD44high cells respond vigorously to H-2Kb immobilized on microspheres and pulsed with peptide, while CD44low cells respond weakly and only at high class I density and peptide concentration. The Ag-presenting surface only has ligands for TCR and CD8 (class I and peptide), thus ruling out the possibility that differences are due to ligand binding by other adhesion or costimulatory receptors that are expressed at high levels on the memory cells. Experiments using anti-TCR mAb as the stimulus and coimmobilized non-Ag class I as a ligand for CD8 suggest that the difference between naive and memory cells may be at the level of stimulation through the TCR. Thus, in addition to expressing increased levels of adhesion receptors that may enhance responses to Ag on APCs, memory CD8+ T cells appear to be intrinsically more sensitive than naive cells to stimulation through the TCR/CD8 complex.

Animals↗

PubMed1997

Artificial cell surface constructs for studying receptor-ligand contributions to lymphocyte activation.

T-cell activation involves interactions between a number of different receptors on the T-cell with their respective ligands on the antigen presenting cell. One approach to studying the contributions of the individual receptors is the use of purified ligands, alone or in combination, to stimulate the cells. However, effective T-cell recognition requires that the ligands be displayed on a surface having dimensions similar to those of a cell. Methods are described for the rapid and efficient immobilization of purified membrane proteins, including class I MHC proteins, B7.1 and ICAM-1, on 5 microm diameter latex microspheres in a manner that preserves biological activity. Non-membrane proteins, such as anti-receptor antibodies, can be immobilized in a similar manner and can be coimmobilized along with membrane proteins. These artificial cell surface constructs can be handled in the same way as cells, including characterization by flow cytometry using antibodies specific for the immobilized proteins and stimulation of T-cell responses. The density of ligand on the surface of the microspheres can be easily varied and controlled and multiple proteins can be immobilized on the same surface. Thus, the described methods provide a great deal of flexibility in assessing the contributions of the various receptors to T-cell signaling and activation, and should be applicable to the study of other cell-cell interactions.

Animals↗

PubMed1997

Migration and activation of antigen-specific CD8+ T cells upon in vivo stimulation with allogeneic tumor.

The response of CD8+ T cells to allogeneic tumor was studied by adoptive transfer of cells from TCR transgenic 2C mice specific for Ld alloantigen. Transferred cells were monitored during the course of a response to i.p. challenge with live P815 (H-2d) using the 1B2 mAb specific for the 2C TCR. Tumor was present in the draining LN and spleen within 3 to 4 days of challenge. The first changes in 1B2+ cells occurred in the spleen on day 4; VLA-4 expression increased in an Ag-specific manner and L-selectin expression decreased in an Ag-nonspecific manner. The number of 1B2+ cells in the spleen declined over days 4 to 6. The first detectable increase in CD25 expression and blast transformation was in the peritoneal cavity beginning days 5 and 6. Clonal expansion was largely limited to this site and was maximal on day 8. As expansion occurred in the peritoneal cavity; the number of 1B2+ cells in the draining LN and spleen also increased. These cells had an activated phenotype (CD44(high), VLA-4(high)) but most did not express CD25 and were not blasts. These results suggest that initial Ag recognition in the spleen results in altered expression of adhesion receptors so that cells gain access to the peritoneal cavity where they undergo clonal expansion and differentiation. Following the response, 1B2+ cells decline in number but a memory population (CD44(high), L-selectin(high and low)) persists for long times in the spleen and LN.

Adoptive Transfer↗

PubMed1997

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells.

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4+ and CD8+ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4+ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8+ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8+ CTL responses. When the dose responses of CD4+ and CD8+ T cells to B7-1 were compared, CD8+ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.

Animals↗

PubMed1997

Stimulation of tumor-specific immunity using tumor cell plasma membrane antigen.

Plasma membranes isolated from tumor cells retain biologically active class I MHC proteins on their surfaces. CD8+ T-cell activation by membrane antigen is much more effective when the small membrane vesicles (<1-microm diameter) are displayed on a surface with dimensions approaching those of a cell (5-microm diameter). Previous work had shown that tumor membrane antigen incorporated onto silica microspheres could augment tumor-specific CTL responses in vivo and significantly reduce syngeneic tumor growth. Antigen on cell-sized solid supports has been termed large multivalent immunogen (LMI). Methods are described for preparing LMI using either silica or latex microspheres. LMI made using either are active in vivo in reducing tumor growth, suggesting that the nature of the support is not critical as long as it is of the appropriate dimensions and has a surface that allows adsorption of the membrane vesicles. Latex microspheres provide some advantages over the previously described silica microspheres with respect to handling and characterization. The effects of LMI on in vivo CTL activation and tumor growth suggest that this approach may have potential for application to clinical immunotherapy of cancers.

Animals↗