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Biomedical subjects

R M Welsh

Publications and source records attributed to R M Welsh.

At least 37 records · Page 2Linked to original sources
PubMed2000

Virus-induced abrogation of transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody.

Treatment with a 2-week course of anti-CD154 antibody and a single transfusion of donor leukocytes (a donor-specific transfusion or DST) permits skin allografts to survive for >100 days in thymectomized mice. As clinical trials of this methodology in humans are contemplated, concern has been expressed that viral infection of graft recipients may disrupt tolerance to the allograft. We report that acute infection with lymphocytic choriomeningitis virus (LCMV) induced allograft rejection in mice treated with DST and anti-CD154 antibody if inoculated shortly after transplantation. Isografts resisted LCMV-induced rejection, and the interferon-inducing agent polyinosinic:polycytidylic acid did not induce allograft rejection, suggesting that the effect of LCMV is not simply a consequence of nonspecific inflammation. Administration of anti-CD8 antibody to engrafted mice delayed LCMV-induced allograft rejection. Pichinde virus also induced acute allograft rejection, but murine cytomegalovirus and vaccinia virus (VV) did not. Injection of LCMV approximately 50 days after tolerance induction and transplantation had minimal effect on subsequent allograft survival. Treatment with DST and anti-CD154 antibody did not interfere with clearance of LCMV, but a normally nonlethal high dose of VV during tolerance induction and transplantation killed graft recipients. We conclude that DST and anti-CD154 antibody induce a tolerant state that can be broken shortly after transplantation by certain viral infections. Clinical application of transplantation tolerance protocols may require patient isolation to facilitate the procedure and to protect recipients.

Animals↗
PubMed2000

Bystander sensitization to activation-induced cell death as a mechanism of virus-induced immune suppression.

Viral infections which induce strong T-cell responses are often characterized by a period of transient immunodeficiency associated with the failure of host T cells to proliferate in response to mitogens or to mount memory recall responses to other antigens. During acute infections, most of the activated, proliferating virus-specific T cells are sensitized to undergo apoptosis on strong T-cell receptor (TCR) stimulation, but it has not been known why memory T cells not specific for the virus fail to proliferate on exposure to their cognate antigen. Using a lymphocytic choriomeningitis virus (LCMV) infection model in which LCMV-immune Thy 1.1(+) splenocytes are adoptively transferred into Thy 1.2(+) LCMV carrier mice, we demonstrate here that T cells clearly defined as not specific for the virus are sensitized to undergo activation-induced cell death on TCR stimulation in vitro. This bystander sensitization was in part dependent on the expression of Fas ligand (FasL) on the activated virus-specific cells and gamma interferon (IFN-gamma) receptor expression on the bystander T cells. We propose that FasL from highly activated antiviral T cells may sensitize IFN-gamma-conditioned T cells not specific for the virus to undergo apoptosis rather than to proliferate on encountering antigen. This may in part explain the failure of memory T cells to respond to recall antigens during acute and persistent viral infections.

Adoptive Transfer↗
PubMed2000

High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection.

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.

Acute Disease↗
PubMed1999

The role of LY49 NK cell subsets in the regulation of murine cytomegalovirus infections.

The distributions and functions of NK cell subsets, as defined by the expression of Ly49 NK cell receptors, were examined in murine CMV (MCMV)-infected mice. MCMV induced a reduction in NK1.1+ cell number in the spleen and an increase in the peritoneal exudate cells. Within the splenic NK1.1+ population, proportional increases in Ly49A+ and Ly49G2+ cells but decreases in Ly49C+ and Ly49D+ cells were observed 3 days post-MCMV infection, but within the peritoneal NK1.1+ cell populations there were proportional decreases in Ly49A+ cells and increases in Ly49C+, Ly49D+, and Ly49G2+ cells. Lymphocytic choriomeningitis virus did not elicit a comparable NK cell subset distribution. Lymphokine-activated killer cells were sorted into different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice. Regulation of MCMV synthesis in these suckling mice was shown to be an IFN-gamma-dependent, perforin- and Cmv-1-independent process, and each NK cell subset mediated anti-viral activity. In adult C57BL/6 mice, the control of MCMV in the spleen is mediated by a perforin-dependent mechanism, regulated in part by the Cmv-1 gene, which maps closely to the Ly49 family. In vivo depletions of either one or two of the Ly49 subsets in adult mice did not affect the ability of the residual NK cells to regulate MCMV synthesis. These data provide evidence of NK cell subset distribution and function in MCMV infection, but no individual subset was required for the Cmv-1-like regulation of MCMV synthesis.

Adoptive Transfer↗
PubMed1999

Attrition of T cell memory: selective loss of LCMV epitope-specific memory CD8 T cells following infections with heterologous viruses.

Using a variety of techniques, including limiting dilution assays (LDA), intracellular IFNgamma assays, and Db-IgG1 MHC dimer staining to measure viral peptide-specific T cell number and function, we show here that heterologous virus infections quantitatively delete and qualitatively alter the memory pool of T cells specific to a previously encountered virus. We also show that a prior history of a virus infection can alter the hierarchy of the immunodominant peptide response to a second virus and that virus infections selectively reactivate memory T cells with distinct specificities to earlier viruses. These results are consistent with a model for the immune system that accommodates memory T cell populations for multiple pathogens over the course of a lifetime.

Animals↗
PubMed1999

Immune deficiency, immune silencing, and clonal exhaustion of T cell responses during viral infections.

Analyses of the complex regulatory networks leading to T cell survival, death, and immune deficiency have been aided in the past year by the dramatic development of new technologies to identify T cells and assess T cell function. These new techniques have shown that functional inactivation and apoptotic elimination of both virus-specific and non-virus-specific T cell populations mold T cell responses to viral infections.

Animals↗
PubMed1998

Stability and diversity of T cell receptor repertoire usage during lymphocytic choriomeningitis virus infection of mice.

Numerous studies have examined T cell receptor (TCR) usage of selected virus-specific T cell clones, yet little information is available regarding the stability and diversity of TCR repertoire usage during viral infections. Here, we analyzed the Vbeta8.1 TCR repertoire directly ex vivo by complementarity-determining region 3 (CDR3) length spectratyping throughout the acute lymphocytic choriomeningitis virus (LCMV) infection, into memory, and under conditions of T cell clonal exhaustion. The Vbeta8 population represented 30-35% of the LCMV-induced CD8(+) T cells and included T cells recognizing several LCMV-encoded peptides, allowing for a comprehensive study of a multiclonal T cell response against a complex antigen. Genetically identical mice generated remarkably different T cell responses, as reflected by different spectratypes and different TCR sequences in same sized spectratype bands; however, a conserved CDR3 motif was found within some same sized bands. This indicated that meaningful studies on the evolution of the T cell repertoire required longitudinal studies within individual mice. Such longitudinal studies with peripheral blood lymphocyte samples showed that (a) the virus-induced T cell repertoire changes little during the apoptosis period after clearance of the viral antigens; (b) the LCMV infection dramatically skews the host T cell repertoire in the memory state; and (c) continuous selection of the T cell repertoire occurs under conditions of persistent infections.

Animals↗
PubMed1998

Protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory T cell populations.

A basic principle of immunology is that prior immunity results in complete protection against a homologous agent. In this study, we show that memory T cells specific to unrelated viruses may alter the host's primary immune response to a second virus. Studies with a panel of heterologous viruses, including lymphocytic choriomeningitis (LCMV), Pichinde (PV), vaccinia (VV), and murine cytomegalo (MCMV) viruses showed that prior immunity with one of these viruses in many cases enhanced clearance of a second unrelated virus early in infection. Such protective immunity was common, but it depended on the virus sequence and was not necessarily reciprocal. Cell transfer studies showed that both CD4 and CD8 T cell populations from LCMV-immune mice were required to transfer protective immunity to naive hosts challenged with PV or VV. In the case of LCMV-immune versus naive mice challenged with VV, there was an enhanced early recruitment of memory phenotype interferon (IFN) gamma-secreting CD4(+) and CD8(+) cells into the peritoneal cavity and increased IFN-gamma levels in this initial site of virus replication. Studies with IFN-gamma receptor knockout mice confirmed a role for IFN-gamma in mediating the protective effect by LCMV-immune T cell populations when mice were challenged with VV but not PV. In some virus sequences memory cell populations, although clearing the challenge virus more rapidly, elicited enhanced IFN-gamma-dependent immunopathogenesis in the form of acute fatty necrosis. These results indicate that how a host responds to an infectious agent is a function of its history of previous infections and their influence on the memory T cell pool.

Adipose Tissue↗
PubMed1998

Detection of a high frequency of virus-specific CD4+ T cells during acute infection with lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV), like many viruses, induces a profound activation and expansion of CD8+ T cells. In contrast, CD4+ T cells do not increase in total number during the acute infection. We show here that mice infected with LCMV have a low but detectable frequency (<1/300) of CD4+ T cells, as detected by IL-2 production in limiting dilution assays, to each of two class II peptides during the peak of the acute LCMV response and into long-term memory. However, during the peak of the acute CD4+ T cell response, >20% of the CD4+ T cells secreted IFN-gamma after stimulation with PMA and ionomycin, and >10% of the CD4+ T cells secreted IFN-gamma after stimulation with the LCMV peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound Ag-specific CD4+ T cell response during viral infections.

Acute Disease↗
PubMed1998

Stability of virus-specific CD4+ T cell frequencies from acute infection into long term memory.

Mice infected with viruses develop long-lasting high frequency memory CD8+ T cell pools, but much less is known about the CD4+ T cell response. FACS analysis revealed the modulation of several activation markers on CD4+ T cells during an acute infection with lymphocytic choriomeningitis virus (LCMV), consistent with an activated cell phenotype. Examination of virus-specific cytokine production using ELISPOT assays showed a significant increase in the number of IFN-gamma-secreting cells in the spleen during an acute LCMV infection. CD8+ T cells made up the majority of the IFN-gamma-producing cells, but analysis of the cell culture supernatants by ELISA showed that the CD4+ T cells produced more IFN-gamma on a per cell basis. Using limiting dilution assays, we examined the CD4+ T cell precursor (Thp) frequency in C57BL/6 mice infected with LCMV. The virus-specific Thp frequency increased from <1/100,000 in uninfected mice to a peak of approximately 1/600 in purified splenic CD4+ T cell populations by 10 days postinfection with LCMV. After the peak of the response, the Thp frequency decreased only about twofold per CD4+ T cell to approximately 1/1200 and remained stable into long term memory. In contrast to the highly activated CD4+ T cells recovered during the acute LCMV infection, the memory CD4+ T cells were maintained at a lower activation state as judged by cell size and ability to secrete IFN-gamma. Thus, like the CD8+ T cell frequencies, the CD4+ T cell frequencies remain elevated after the acute infection subsides and stay elevated throughout long term immunity.

Acute Disease↗
PubMed1998

Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes.

Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70-peptide mixture and challenged with LCMV. Virus titers were reduced 10-100-fold in these mice compared to control mice. Immunization with the hsp70-peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.

Adjuvants, Immunologic↗
PubMed1998

T-cell-independent antiviral antibody responses.

Recent work has shown that viruses can act in vivo as T-cell-independent antigens, eliciting protective, isotype-switched antibodies in the absence of conventional TCR alpha beta+ T cell help. Inactivated virus or virus-like particles can stimulate IgM production, but factors induced during live virus infection appear to be required to induce the isotype switch that leads to IgG or IgA responses.

Animals↗
PubMed1998

Apoptotic regulation of T cells and absence of immune deficiency in virus-infected gamma interferon receptor knockout mice.

Acute viral infections often induce a transient period of immune deficiency in which the host's T cells fail to proliferate in response to T-cell mitogens and fail to make an antigen-specific memory recall response. This has been associated with the enhanced sensitivity of these highly activated T cells to undergo apoptosis, or activation-induced cell death (AICD), upon T-cell receptor ligation. Here we show that gamma interferon receptor-deficient (IFN-gamma R-/-) mice mount a T-cell response to lymphocytic choriomeningitis virus (LCMV) infection but fail to undergo the transient immune deficiency. Instead, their T cells were hyperproliferative and relatively, but not completely, resistant to AICD. The immune response returned to homeostasis, but with delayed kinetics, in parallel with delayed clearance of the virus. Wild-type mice receiving high doses of disseminating LCMV Clone 13 are known to undergo clonal exhaustion of their virus-specific cytotoxic T lymphocytes (CTL). To determine whether this process was mediated by AICD associated with IFN-gamma or with Fas-Fas ligand interactions, LCMV-specific precursor CTL frequencies were examined in LCMV Clone 13-infected IFN-gamma R-/- or lpr (Fas-deficient) mice. In both instances, viral persistence was established and CTL precursors were greatly eliminated. This finding indicates that clonal exhaustion of CTL does not require IFN-gamma or Fas, even though both molecules influence AICD and the transient immune deficiency seen in the LCMV infection.

Animals↗
PubMed1998

Evaluation of the Galalpha1-3Gal epitope as a host modification factor eliciting natural humoral immunity to enveloped viruses.

Human sera contain high levels of natural antibody (Ab) to Galalpha1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding alpha-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galalpha1-3Gal and was blocked by incorporation of soluble Galalpha1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galalpha1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galalpha1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galalpha1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galalpha1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.

Animals↗
PubMed1998

T-Cell-independent immunoglobulin G responses in vivo are elicited by live-virus infection but not by immunization with viral proteins or virus-like particles.

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor beta chain [TCR-beta] -/- or TCR-betaxdelta -/-) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.

Animals↗
PubMed1997

Cytolytically active memory CTL present in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection.

Generally, it has been assumed that memory T cells are dormant and inactive cells in the absence of their specific Ag. Recent work has challenged this assumption by showing that a portion of the CD8+ memory T cell pool is in cycle. In this study, we demonstrate that a significant number of blast-size memory CD8+ T cells in mice, long after lymphocytic choriomeningitis virus (LCMV) infection, mediate cytolysis against highly sensitive targets without any in vivo or in vitro restimulation and expansion with Ag. Peptide-coated RMA-S targets were sufficiently sensitive to detect low but significant cytolytic activity in bulk 51Cr release assays in nonstimulated LCMV-specific splenic memory CTL populations. Most of the directly cytotoxic activity was against the GP33 epitope, and this persisted throughout the lifetime of the mouse following infection. The cytotoxic activity was not inhibited by cyclosporin A, indicating that these cells were already in an active state and not dependent on further stimulation in vitro. It was formally shown that the cytotoxic activity was mediated by the CD8+ CTL by sorting for the blast-size CD8+ population and by blocking target cell lysis with anti-CD8 Ab. Thus, at any time after the original infection some portion of the memory CD8+ T cell pool is cycling, and it remains cytolytically active long after resolution of the original infection. These CTL may provide a rapidly acting defense mechanism against reinfection.

Animals↗
PubMed1997

Minimal bystander activation of CD8 T cells during the virus-induced polyclonal T cell response.

Acute infections with viruses such as lymphocytic choriomeningitis virus (LCMV) are associated with a massive polyclonal T cell response, but the specificities of only a small percentage of these activated T cells are known. To determine if bystander stimulation of T cells not specific to the virus plays a role in this T cell response, we examined two different systems, HY-specific T cell receptor (TCR)-transgenic mice, which have a restricted TCR repertoire, and LCMV-carrier mice, which are tolerant to LCMV. LCMV infection of HY-transgenic C57BL/6 mice induced antiviral CTLs that lysed target cells coated with two of the three immunodominant epitopes previously defined for LCMV (glycoprotein 33 and nucleoprotein 397). Although LCMV-induced cytotoxic T lymphocytes (CTLs) from C57BL/6 mice could lyse uninfected H-2(k) and H-2(d) allogeneic targets, LCMV-induced CTLs from HY mice lysed only the H-2(k)-expressing cells. The HY mice generated both anti-H-2(k) and anti-H-2(d) CTL in mixed leukocyte reactions, providing evidence that the generation of allospecific CTLs during acute LCMV infection is antigen specific. During the LCMV infection there was blastogenesis of the CD8+ T cell population, but the HY-specific T cells (as determined by expression of the TCR-alpha chain) remained small in size. To examine the potential for bystander stimulation under conditions of a very strong CTL response, T cell chimeras were made between normal and HY mice. Even in the context of a normal virus-induced CTL response, no stimulation of HY-specific T cells was observed, and HY-specific cells were diluted in number by day 9 after infection. In LCMV-carrier mice in which donor and host T cells could be distinguished by Thy1 allotypic markers, adoptive transfer of LCMV-immune T cells into LCMV-carrier mice, whose T cells were tolerant to LCMV, resulted in activation and proliferation of donor CD8 cells, but little or no activation of host CD8 cells. These results support the hypothesis that the massive polyclonal CTL response to LCMV infection is virus-specific and that bystander activation of non-virus-specific T cells is not a significant component of this response.

Animals↗