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J Sprent

Publications and source records attributed to J Sprent.

At least 19 recordsLinked to original sources
PubMed1999

Strong TCR ligation without costimulation causes rapid onset of Fas-dependent apoptosis of naive murine CD4+ T cells.

Activation-induced cell death of T cells typically occurs late in the primary response after a prior proliferative response. Here, we describe a novel form of cell death in which purified naive murine CD4+ cells undergo apoptosis within 18 h in vitro after strong TCR ligation. Such rapid-onset TCR-mediated death of T cells does not involve cell division and is Fas-dependent, inhibited by CD28 (and IL-6) costimulation and enhanced by IL-4 and IL-7; by contrast, spontaneous death of CD4+ cells cultured alone is Fas-independent and inhibited by IL-4 and IL-7. TCR-mediated Fas-dependent death of CD4+ cells is prevented by combined TCR/Fas ligation and by drugs that inhibit calcineurin-dependent signaling and mitogen-activated protein kinase MEK1 activation.

Animals↗
PubMed1999

Several different cell surface molecules control negative selection of medullary thymocytes.

Repeated attempts to show that costimulation for negative selection is controlled by a single cell surface molecule have been unsuccessful. Thus, negative selection may involve multiple cell surface molecules acting in consort. In support of this idea, we show here that at least three cell surface molecules, namely CD28, CD5, and CD43, contribute to Fas-independent negative selection of the tolerance-susceptible population of heat-stable antigen (HSA)hiCD4+8- cells found in the medulla. The costimulatory function of these three molecules can be blocked by certain cytokines, IL-4 and IL-7, and coinjecting these cytokines with antigen in vivo abolishes negative selection; Fas-dependent negative selection, however, is maintained. The results suggest that efficient negative selection requires the combined functions of at least four cell surface molecules: CD28, CD5, CD43, and Fas.

Animals↗
PubMed1999

T-cell turnover in vivo and the role of cytokines.

In addition to responding to specific antigen, CD8+ T-cells with a memory (CD44hi) phenotype undergo bystander proliferation when exposed to certain cytokines, notably type I interferons (IFN I), in vivo; such proliferation does not require T-cell receptor ligation. Since IFN I is unable to induce proliferation of purified CD44hi CD8+ cells in vitro, stimulation of these cells in vivo may reflect IFN I-dependent release of other cytokines. Evidence is presented that IFN I induces macrophages to synthesize IL-15 mRNA and that, at the protein level, IL-15 causes selective stimulation of CD44hi CD8+ (but not CD4+) cells, both in vitro and in vivo. This finding raises the possibility that synthesis of IL-15 during infection may induce bystander proliferation of memory-phenotype CD8+ cells.

Animals↗
PubMed1999

Thymic selection by a single MHC/peptide ligand: autoreactive T cells are low-affinity cells.

In H2-M- mice, the presence of a single peptide, CLIP, bound to MHC class II molecules generates a diverse repertoire of CD4+ cells. In these mice, typical self-peptides are not bound to class II molecules, with the result that a very high proportion of H2-M- CD4+ cells are responsive to the various peptides displayed on normal MHC-compatible APC. We show here, however, that such "self" reactivity is controlled by low-affinity CD4+ cells. These cells give spectacularly high proliferative responses but are virtually unreactive in certain other assays, e.g., skin graft rejection; responses to MHC alloantigens, by contrast, are intense in all assays. Possible explanations for why thymic selection directed to a single peptide curtails self specificity without affecting alloreactivity are discussed.

Adoptive Transfer↗
PubMed1998

Type I interferon-mediated stimulation of T cells by CpG DNA.

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR-/- mice. Second, in contrast to normal T cells, the failure of purified IFN-IR-/- T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-gamma) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR-/-) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.

Animals↗
PubMed1998

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice.

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.

Adenocarcinoma↗
PubMed1998

Requirements for stimulating naive CD8+ T cells via signal 1 alone.

In the absence of costimulation, TCR recognition of peptide/MHC complexes is generally considered to be nonimmunogenic. In agreement with this view, naive TCR transgenic CD8+ cells failed to respond to specific peptides presented by MHC class I (Ld) molecules bound to mouse RBC. However, peptide/Ld complexes presented by cell-sized beads or bound to plastic led to overt proliferative responses in the absence of added cytokines. Significantly, equivalent strong proliferative responses occurred when mouse RBC were fixed with glutaraldehyde before Ld coupling. The implication therefore is that the intensity of signaling via the TCR is a reflection of the mobility of the ligand being recognized; TCR signaling is weak when the ligand can move laterally on the cell membrane but strong when the ligand is immobilized.

Animals↗
PubMed1998

Anti-viral immunity: spotting virus-specific T cells.

Historically, quantitation of virus-specific CD8+ T cells has been accomplished by limiting dilution analysis of cytotoxic precursor cells. Recent studies have shown that this technique greatly underestimates the actual number of antigen-specific cells and have provided new insight into anti-viral immune responses.

Animals↗
PubMed1998

Bystander stimulation of T cells in vivo by cytokines.

Immune responses to infectious agents, especially viruses, are often associated with extensive proliferation of T cells and transient enlargement of the lymphoid tissues. Since the precursor frequency of T cells for specific antigen is low, the bulk of the T cells proliferating in the primary response are presumably stimulated via non-antigen-specific mechanisms, e.g. via cytokines elicited by the infectious agent concerned. Such 'bystander' stimulation of T cells occurs in mice injected with agents that elicit production of type I interferon (IFN I). Induction of IFN I in vivo causes marked stimulation of the CD44hi subset of CD8+ T cells and is prominent after injection of live viruses or products of bacteria such as lipopolysaccharide. Cytokines elicited by infectious agents may act as adjuvants during the primary response and could serve to boost the survival of long-lived memory cells.

Adjuvants, Immunologic↗
PubMed1998

A role for Fas in negative selection of thymocytes in vivo.

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (</=1 microg) but not by high doses (100 microg). Similar findings applied when T cell receptor transgenic mice were injected with specific peptide. These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.

Amino Acid Sequence↗
PubMed1998

DNA as an adjuvant: capacity of insect DNA and synthetic oligodeoxynucleotides to augment T cell responses to specific antigen.

How strong adjuvants such as complete Freund's adjuvant (CFA) promote T cell priming to protein antigens in vivo is still unclear. Since the unmethylated CpG motifs in DNA of bacteria and other nonvertebrates are stimulatory for B cells and antigen-presenting cells, the strong adjuvanticity of CFA could be attributed, at least in part, to the presence of dead bacteria, i.e., a source of stimulatory DNA. In support of this possibility, evidence is presented that insect DNA in mineral oil has even stronger adjuvant activity than CFA by a number of parameters. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs mimic the effects of insect DNA and, even in soluble form, ODNs markedly potentiate clonal expansion of T cell receptor transgenic T cells responding to specific peptide.

Adjuvants, Immunologic↗
PubMed1998

Lifespan of gamma/delta T cells.

Information on the turnover and lifespan of murine gamma/delta cells was obtained by administering the DNA precursor, bromodeoxyuridine (BrdU), in the drinking water and staining lymphoid cells for BrdU incorporation. For TCR-gamma/delta (Vgamma2) transgenic mice, nearly all gamma/delta thymocytes became BrdU+ within 2 d and were released rapidly into the peripheral lymphoid tissues. These recent thymic emigrants (RTEs) underwent phenotypic maturation in the periphery for several days, but most of these cells died within 4 wk. In adult thymectomized (ATx) transgenic mice, only a small proportion of gamma/delta cells survived as long-lived cells; most of these cells had a slow turnover and retained a naive phenotype. As in transgenic mice, the majority of RTEs generated in normal mice (C57BL/6) appeared to have a restricted lifespan as naive cells. However, in marked contrast to TCR transgenic mice, most of the gamma/delta cells surviving in ATx normal mice had a rapid turnover and displayed an activated/memory phenotype, implying a chronic response to environmental antigens. Hence, in normal mice many gamma/delta RTEs did not die but switched to memory cells.

Animals↗
PubMed1998

Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15.

Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.

Animals↗
PubMed1998

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells.

Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.

Animals↗
PubMed1998

CD40 ligand-mediated interactions are involved in the generation of memory CD8(+) cytotoxic T lymphocytes (CTL) but are not required for the maintenance of CTL memory following virus infection.

CD8(+) cytotoxic T lymphocytes (CTL) play a key role in the control of many virus infections, and the need for vaccines to elicit strong CD8(+) T-cell responses in order to provide optimal protection in such infections is increasingly apparent. However, the mechanisms involved in the induction and maintenance of CD8(+) CTL memory are currently poorly understood. In this study, we investigated the involvement of CD40 ligand (CD40L)-mediated interactions in these processes by analyzing the memory CTL response of CD40L-deficient mice following infection with lymphocytic choriomeningitis virus (LCMV). The maintenance of memory CD8(+) CTL precursors (CTLp) at stable frequencies over time was not impaired in CD40L-deficient mice. By contrast, the initial generation of memory CTLp was affected. CD40L-deficient mice produced lower levels of CD8(+) CTLp during the primary immune response to LCMV than did wild-type controls, despite the fact that the LCMV-specific effector CTL response of CD40L-deficient mice was indistinguishable from that of control animals. The differentiation of naïve CD8(+) T cells into effector and memory CTL thus involves pathways that can be discriminated from each other by their requirement for CD40L-mediated interactions. Expression of CD40L by CTLp themselves was not an essential step during their expansion and differentiation from naïve CD8(+) cells into memory CTLp; instead, the reduction in memory CTLp generation in CD40L-deficient mice was likely a consequence of defects in the CD4(+) T-cell response mounted by these animals. These results thus suggest a previously unappreciated role for CD40L in the generation of CD8(+) memory CTLp, the probable nature of which is discussed.

Animals↗
PubMed1997

Mitogenicity of DNA from different organisms for murine B cells.

Recent evidence that DNA from bacteria causes polyclonal activation of mouse B cells raises the question of whether DNA from other organisms has similar properties. Extending prior studies on bacteria and insects, we show here that the capacity of DNA to stimulate B cells correlates closely with hypomethylation of DNA CpG dinucleotide motifs. Thus strong stimulation of B cells was seen with DNA from various organisms displaying little or no methylation of CpG motifs, i.e., yeast, nematodes, and molluscs in addition to bacteria and insects. For these organisms, DNA induced nearly all B cells (including small resting B cells) to up-regulate the activation marker, CD69, and caused many B cells to enter the cell cycle, indicative of polyclonal activation; this effect was not seen after selective methylation of CpG motifs (tested on yeast DNA). By contrast, no stimulation of B cells was seen with DNA from organisms whose CpG motifs are heavily methylated, i.e., various vertebrates (mammals, fish, and frogs) and plants (corn). Despite this correlation, DNA prepared from two murine cell lines exhibiting hypomethylation of CpG motifs caused little or no stimulation of B cells. Thus, the idea that the stimulatory properties of DNA correlate solely with the presence of unmethylated CpG motifs may be an oversimplification.

Animals↗
PubMed1997

T cell stimulation in vivo by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) from gram-negative bacteria causes polyclonal activation of B cells and stimulation of macrophages and other APC. We show here that, under in vivo conditions, LPS also induces strong stimulation of T cells. As manifested by CD69 upregulation, LPS injection stimulates both CD4 and CD8(+) T cells, and, at high doses, stimulates naive (CD44(lo)) cells as well as memory (CD44(hi)) cells. However, in terms of cell division, the response of T cells after LPS injection is limited to the CD44(hi) subset of CD8(+) cells. In contrast with B cells, proliferative responses of CD44(hi) CD8(+) cells require only very low doses of LPS (10 ng). Based on studies with LPS-nonresponder and gene-knockout mice, LPS-induced proliferation of CD44(hi) CD8(+) cells appears to operate via an indirect pathway involving LPS stimulation of APC and release of type I (alpha, beta) interferon (IFN-I). Similar selective stimulation of CD44(hi) CD8(+) cells occurs in viral infections and after injection of IFN-I, implying a common mechanism. Hence, intermittent exposure to pathogens (gram-negative bacteria and viruses) could contribute to the high background proliferation of memory-phenotype CD8(+) cells found in normal animals.

Animals↗