Biomedical subjects

H Pircher

Publications and source records attributed to H Pircher.

At least 37 records · Page 2Linked to original sources

PubMed1999

Distinct migration patterns of naive and effector CD8 T cells in the spleen: correlation with CCR7 receptor expression and chemokine reactivity.

Changes in the migration pattern of lymphocytes represent a key event in the evolution of an immune response since they enable lymphocytes to gain access to infected tissues. We studied the location of virus-specific CD8 T cells in various splenic compartments in response to infection with lymphocytic choriomeningitis virus (LCMV), either in situ or by adoptive cell transfers using T cells from transgenic (tg) mice expressing an LCMV-specific TCR. Naive tg T cells were predominantly localized in the periarteriolar lymphoid sheath, where they proliferated extensively after virus infection. In contrast, in vivo activated effector T cells failed to enter white pulp areas and accumulated in the red pulp. The different homing patterns of naive and effector CD8 T cells in vivo correlated well with their CCR7 chemokine receptor expression and their reactivity to the secondary lymphoid tissue chemokine (SLC). Thus, down-regulation of CCR7 expression on CD8 effector T cells rendered them unre sponsive to SLC, which controls T cell homing into white pulp of spleen and lymph nodes. Exclusion of CD8 effector T cells from these sites may represent an important mechanism to protect professional antigen-presenting cells from cytotoxic T cell attack and thus to prevent a prematuredecline of the immune response.

Animals↗

PubMed1999

Identification of an alpha2,6-sialyltransferase induced early after lymphocyte activation.

We have used mRNA differential display PCR to search for genes induced in activated T cells and we identified a gene encoding an alpha2,6-sialyltransferase (ST6GalNAc IV) that is rapidly induced in lymphocytes after antigen or mitogen stimulation. The 3.6 kb full-length cDNA clone (MK45) obtained contained a single open reading frame encoding a 302 amino acid protein and a 2.5 kb 3' untranslated region. MK45 expression in in vivo-activated CD8 T cells reached the highest level 4 h after antigen triggering and then declined rapidly to nearly base levels within 45 h. Northern blot analysis further revealed that MK45 expression was also induced in LPS-activated B cells and antigen-triggered CD4 T cells in vitro. MK45 expression was low or undetectable in most other mouse tissues examined, when compared to activated lymphocytes. Importantly, the mRNA expression level of other sialyltransferases remained largely unchanged during the early stage of lymphocyte activation. Finally, increased ecto-sialyltransferase activity and an altered sialylation pattern were demonstrated on the cell surface of early activated CD8 T cells. Our report identifies a candidate sialyltransferase gene that is involved in the early alteration of the sialylation pattern of cell surface molecules in activated lymphocytes.

Amino Acid Sequence↗

PubMed1998

Virus-activated CD8 T cells and lymphokine-activated NK cells express the mast cell function-associated antigen, an inhibitory C-type lectin.

The mast cell function-associated Ag (MAFA) is an inhibitory C-type lectin that was originally identified on the cell surface of a rat mucosal mast cell line, RBL-2H3. We have cloned the mouse homologue of the rat MAFA gene, and Northern blot analysis revealed that mouse MAFA (mMAFA) gene expression was strongly induced in effector CD8 T cells and lymphokine-activated NK cells but not in effector CD4 T cells and in mouse mast cells. Moreover, mMAFA gene expression was only found in effector CD8 T cells that had been primed in vivo with live virus because in vitro activated CD8 T cells did not express mMAFA. Primary sequence comparison revealed a high degree of conservation (89% similarity) between rat MAFA and mMAFA. Thus, the MAFA molecule in the mouse is a putative inhibitory receptor on anti-viral CD8 T cells induced in vivo and on NK cells.

Amino Acid Sequence↗

PubMed1998

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo.

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.

Adoptive Transfer↗

PubMed1998

Viral and bacterial infections interfere with peripheral tolerance induction and activate CD8+ T cells to cause immunopathology.

We studied the impact of various infectious and proinflammatory agents on the induction of peripheral T cell tolerance. Adoptive transfer of CD8+ T cells from lymphocytic choriomeningitis virus (LCMV) T cell receptor transgenic mice into LCMV antigen transgenic mice expressing the LCMV glycoprotein epitope (gp) 33-41 under control of a major histocompatibility complex class I promoter led to efficient induction of peripheral tolerance after a period of transient activation. If, however, the recipient mice were challenged with viral or bacterial infections or proinflammatory agents (lipopolysaccharide or Poly:IC) early after cell transfer, tolerance induction was prevented and instead, CD8+ T cell activation leading to vigorous expansion and generation of cytolytic activity ensued. This became manifest in significant immunopathology mainly involving destruction of the splenic architecture and lysis of antigen-expressing lymphocyte and macrophage populations. Important parameters involved in the activation of host-reactive T cells by nonspecific infectious agents included the presence, localization, and quantity of the specific transgene-encoded self-antigen; in contrast, CD4+ T cells were not required. In mice surviving the acute phase, the transferred CD8+ T cells persisted at high levels in an anergic state; they were unable to generate cytolytic activity in vitro or to control LCMV infection in vivo. These results impinge on our understanding of the role of infectious agents in graft verus host reactions towards minor histocompatibility antigens.

Animals↗

PubMed1998

Constitutive expression of Bcl-xL or Bcl-2 prevents peptide antigen-induced T cell deletion but does not influence T cell homeostasis after a viral infection.

We examined the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) in mice doubly transgenic for an LCMV-specific TCR and for either bcl-xL or bcl-2. Clonal down-sizing of the anti-viral CD8+ T cell response and the generation of T cell memory was not influenced by constitutive expression of these anti-apoptotic proteins in T cells. Expression of Bcl-xL or Bcl-2 did, however, prevent LCMV peptide-induced peripheral deletion of mature CD8+ T cells in vivo and apoptosis of activated LCMV-specific effector T cells in vitro. The CD8+ T cells "rescued" by Bcl-xL or Bcl-2 from peptide antigen-induced cell death were anergic and this could not be reversed by addition of IL-2 in vitro or by adoptive transfer into antigen-free recipient mice followed by LCMV infection in vivo. Taken together, we show here that 1) Bcl-xL or Bcl-2 are functionally equivalent in their ability to modulate CD8+ T cell survival in vivo, 2) distinct apoptosis signaling pathways exist in CD8+ T cells, one that can be inhibited by Bcl-2 or Bcl-xL and one that cannot be blocked, and 3) apoptosis of CD8+ effector T cells during the declining phase of an immune response is not prevented by constitutive expression of the anti-apoptotic proteins Bcl-xL and Bcl-2.

Animals↗

PubMed1998

Beta-galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells.

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a beta-galactoside-binding protein (betaGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8+ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8+ T cells. LGALS1 expression could be induced in CD4+ and CD8+ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric betaGBP was released by activated CD8+ T cells and in vitro experiments further showed that recombinant betaGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8+ T cells. Thus, these data indicate a role of betaGBP as an autocrine negative growth factor for CD8+ T cells.

Animals↗

PubMed1998

Antigen persistence and time of T-cell tolerization determine the efficacy of tolerization protocols for prevention of skin graft rejection.

We studied antigen-specific T-cell tolerization therapy using skin transplantation across a defined minor histocompatibility antigen difference. Specific tolerization protocols using short-lived peptide or long-lived spleen cells presenting the peptide as antigen prevented graft rejection without immunosuppression when started before or as long as 10 days after transplantation. Peptide-induced T-cell tolerance was transient, and antigen presentation by the graft was not sufficient to maintain tolerance. In contrast, transfer of antigen-expressing lymphoid cells induced long-lasting tolerance correlating with donor cell chimerism. These findings show that antigen-specific tolerization can induce graft acceptance even when begun after transplantation and that long-term graft survival depends on persistence of the tolerizing antigen.

Animals↗

PubMed1998

An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

Animals↗

PubMed1997

Alterations in the level of CD45 surface expression affect the outcome of thymic selection.

CD45 is a receptor protein tyrosine phosphatase whose activity is required for thymocyte development and TCR-mediated signal transduction. Here we show that positive selection of TCR-alphabeta transgenic thymocytes is completely blocked in CD45 exon 6 -/- gene-deficient (CD45 -/-) mice that express the P14 TCR specific for the lymphocytic choriomeningitis virus. Thymocytes from mice heterozygous for the targeted disruption of the CD45 gene (CD45 +/-) displayed a reduction in both CD45 surface intensity and enzymatic CD45 protein tyrosine phosphatase activity. Surprisingly, positive thymocyte selection was enhanced in CD45 +/- mice as characterized by an up-regulation of the P14 TCR on thymocytes and increased numbers of transgenic T cells. Using a variant of lymphocytic choriomeningitis virus that is impaired in the induction of negative selection of P14 thymocytes, we also show that the reduction of CD45 surface expression in CD45 +/- mice rendered P14 transgene thymocytes susceptible to negative selection. These data demonstrate that changes in the expression level of the receptor protein tyrosine phosphatase CD45 can alter thymic selection.

Animals↗

PubMed1997

Differentiation of CD4(high)CD8(low) coreceptor-skewed thymocytes into mature CD8 single-positive cells independent of MHC class I recognition.

Thymocytes with a CD4(hi)CD8(lo) coreceptor-skewed (CRS) phenotype have been shown to contain precursors for CD8 single-positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4(hi)CD8(lo) cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I-restricted P14 T cell receptor (TCR), on the H-2bm13 background, generate a large number of CD4(hi)CD8(lo) CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4(hi)CD8(lo) population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up-regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re-expressed CD8 but failed to re-express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide-pulsed antigen-presenting cells. Our data suggest that CD4(hi)CD8(lo) CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.

Animals↗

PubMed1997

Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology.

Peptide-specific down-regulation of T cell responses may represent a powerful tool to intervene in autoimmune diseases or graft rejections. It is therefore important to know whether peptide treatment tolerizes both naive and antigen-experienced memory T lymphocytes. Here we show that a major histocompatibility complex class I binding peptide, derived from the glycoprotein (GP33 peptide) of lymphocytic choriomeningitis virus (LCMV), specifically tolerized naive cytotoxic T lymphocytes (CTL) when administered three times intraperitoneally in incomplete Freund's adjuvants. However, in the presence of GP33-specific memory CTL in LCMV-primed mice, the same treatment had a general immunosuppressive effect on unrelated third-party antigen-specific T cell responses and caused severe immunopathological damage to the spleen.

Adoptive Transfer↗

PubMed1996

IL-4 differentiates naive CD8+ T cells to a "Th2-like" phenotype: a link between viral infections and bronchial asthma.

Viral infections of the lung have been postulated to be a major factor in the etiology of bronchial asthma, a disease characterized by eosinophilic inflammation of the airways. In addition, upper respiratory tract infection in asthmatic individuals results in an exacerbation of the disease. Nevertheless, the mechanisms by which viral infection leads to disease exacerbation are poorly understood. CD8+ T cells play an important role in the host defense responses against viral infection, although to date, there are no reports to suggest that CD8+ T cells play any role in eosinophil recruitment. In the present study, we report that CD8+ T cells activated by either immobilized CD3 mAb or specific antigen can switch to a phenotype that produces Th2 cytokines and secretes less IFN-gamma. Moreover, in vivo, if a lung mucosal Th2 immune response exists, then antigen-specific activation of CD8 cells results in the development of lung eosinophilic inflammation mediated by the secretion of IL-5 from CD8+ T cells. These results may explain the link between viral infections and bronchial asthma, as this IL-4-dependent switch to CD8+ T cells to IL-5 secretion may not only exacerbate asthma by recruiting eosinophils into the lungs, but the impaired IFN-gamma production may also lead to delayed viral clearance.

Animals↗

PubMed1996

On the role of antigen in maintaining cytotoxic T-cell memory.

This study evaluated whether T-cell memory reflects increased precursor frequencies of specific long-lived T cells and/or a low-level immune response against some form of persistent antigen. Antivirally protective CD8+ T-cell memory was analyzed mostly in the original vaccinated host to assess the role of antigen in its maintenance. T-cell mediated resistance against reinfection was measured in the spleen and in peripheral solid organs with protocols that excluded protection by antibodies. In vivo protection was compared with detectable cytotoxic T-lymphocyte precursor frequencies determined in vitro. In the spleen, in vitro detectable cytotoxic T-lymphocyte precursor frequencies remained stable independently of antigen, conferring resistance against viral replication in the spleen during reinfection. In contrast, T-cell mediated resistance against reinfection of peripheral solid organs faded away in an antigen-dependent fashion within a few days or weeks. We show that only memory T cells persistently or freshly activated with antigen efficiently extravasate into peripheral organs, where cytotoxic T lymphocytes must be able to exert effector function immediately; both the capacity to extravasate and to rapidly exert effector function critically depend on restimulation by antigen. Our experiments document that the duration of T-cell memory protective against peripheral reinfection depended on the antigen dose used for immunization, was prolonged when additional antigen was provided, and was abrogated after removal of antigen. We conclude that T-cell mediated protective immunity against the usual peripheral routes of reinfection is antigen-dependent.

Animals↗

PubMed1996

Visualization, characterization, and turnover of CD8+ memory T cells in virus-infected hosts.

The cellular basis of T cell memory is a controversial issue and progress has been hampered by the inability to induce and to trace long-term memory T cells specific for a defined antigen in vivo. By using the murine model of lymphocytic choriomeningitis virus (LCMV) infection and an adoptive transfer system with CD8+ T cells from transgenic mice expressing an LCMV-specific T cell receptor, a population of authentic memory T cells specific for LCMV was generated and analyzed in vivo. The transgenic T cells that have expanded (1,000-fold) and then decreased (10-fold) in LCMV-infected C57BL/6 recipient mice exhibited the following characteristics: they were (a) of larger average cell size than their naive counterparts but smaller than day 8 effector cells; (b) heterogeneous with respect to expression of cell surface "memory" markers; and (c) directly cytolytic when isolated from recipient spleens. The time-dependent proliferative activity of these LCMV-specific memory T cells was analyzed in the recipients by bromodeoxyuridine labeling experiments in vivo. The experiments revealed that LCMV-specific CD8+ memory T cells can persist in LCMV-immune mice for extended periods of time (>2 mo) in the absence of cell division; the memory population as a whole survived beyond 11 mo.

Amino Acid Sequence↗

PubMed1996

Homeostatic regulation of CD8+ T cells after antigen challenge in the absence of Fas (CD95).

The role of Fas in the homeostatic regulation of CD8+ T cells after antigen challenge was analyzed in the murine model of lymphocytic choriomeningitis virus (LCMV) infection. Mice homozygous for the lpr mutation and carrying T cell receptor (TCR) alphabeta transgenes specific for the LCMV glycoprotein peptide aa 33-41 in the context of H-2Db were used. Five main results emerged: first, development of lymphadenopathy and of CD4- CD8- double-negative B220+ T cells in lpr mice was not inhibited by the alphabeta TCR transgenes; second, tolerance induction and peripheral deletion of CD8+ T cells induced by LCMV glycoprotein peptide injection was independent of Fas expression; third, clonal down-regulation of Fas-deficient TCR-transgenic CD8+ T cells after acute LCM virus infection was identical to the decline of transgenic T cells expressing Fas; fourth, in vivo activated CD8+ effector T cells from TCR transgenic and TCR-lpr/lpr mice were equally susceptible to activation-induced cell death in vitro; and fifth, transgenic effector T cells from lpr/lpr mice were cleared in the declining phase of the immune response in vivo without giving rise to CD4- CD8- double-negative T cells. Taken together, these data suggest that the homeostatic regulation of CD8+ T cells after antigen challenge in vivo is regulated by mechanisms that do not require Fas.

Animals↗

PubMed1996

A novel mouse thymocyte antigen (F3Ag): down-regulation during the CD4+CD8+ double-positive stage indicates positive selection.

We describe a novel mAb (F3) which reacts with a 65 kDa thymocyte surface protein, expressed on approximately 80% of thymocytes, referred to as F3Ag. In ontogeny, F3Ag expression begins in the CD4(-)CD8(-) double-negative (DN) CD25(+) population and is maintained through approximately 85% of the CD4(+)CD8(+) double-positive (DP) stage. DP cells with high TCR expression and CD4(+) single-positive (SP) cells are predominantly negative for F3Ag, whereas many CD8(+) SP thymocytes express F3Ag. F3Ag-DP thymocytes show a reduced expression of RAG-1 and RAG-2 compared with F3Ag+ DP cells. The shutdown of F3Ag expression during the DP stage is related to positive selection: mice deficient for MHC class I and class II molecules maintain F3Ag expression in almost all DP cells. Transgenic (tg) mice carrying TCR restricted for MHC class II show a more pronounced down-regulation of F3Ag in the DP compartment than normal mice, depending on the presence of a positively selecting MHC. The size of the F3Ag- DP subset is positively correlated with the efficacy of positive selection into the CD4(+) SP compartment. Because some CD8(+) SP cells express F3Ag, the relationship between F3Ag down-regulation and positive selection is less obvious in DP cells of mice carrying MHC class I-restricted tg TCR. However, in reaggregate thymic organ cultures, sorted F3Ag- DP cells differentiate into CD8(+) SP cells more rapidly than do F3Ag+ DP cells. Thus, after down-regulation in the DP stage, a proportion of CD8(+) SP cells appears to re-express F3Ag. In addition, the proportion of F3Ag-CD8(+) SP cells depends on the efficacy of positive selection into the CD8 lineage. Taken together, the regulation of the expression of the F3Ag appears to be associated with signals that control thymic repertoire selection.

Animals↗

PubMed1996

On T cell memory: arguments for antigen dependence.

Memory is a hallmark of the immune system. Considerable progress has been made towards understanding B cell memory, but T cell memory remains poorly understood and its nature is controversial. There is good evidence that B cell memory is driven by antigen, but the antigen dependence of T cell memory is still being debated. For several years we have investigated the nature, duration and antigen dependence of different aspects of CD8+ T cell memory and this review will discuss our findings as well as how and why they differ from some other results. As others, we find that antigen, due to proliferation of antigen-specific T cell clones, induces a shift in the T cell repertoire which remains detectable for years as an elevated cytotoxic T cell precursor frequency (CTLp) in lymphoid organs. Also in the absence of antigen, in vitro assays for T cell memory which invariably isolate memory T cells from these lymphoid organs therefore remain positive. In contrast, immunity against reinfection with a pathogen requires more than just elevated numbers of CTLp in lymphoid organs. Since reinfection usually takes place via peripheral nonlymphoid tissue, these CTLp have to a) efficiently extravasate and patrol through such tissues, and b) be immediately able to exert effector function in case of reinfection. Both functions, require a certain level of activation which critically depends on T cell stimulation by persisting antigen.

Animals↗