Search PubMed⌕ Search

Biomedical subjects

P C Doherty

Publications and source records attributed to P C Doherty.

At least 127 records · Page 7Linked to original sources
PubMed1994

Protection against lethal lymphocytic choriomeningitis virus (LCMV) infection by immunization of mice with an influenza virus containing an LCMV epitope recognized by cytotoxic T lymphocytes.

The reverse genetics system has made it possible to modify the influenza virus genome. By this method, we were able to assess influenza virus as a vaccine vector for protecting BALB/c mice against otherwise lethal lymphocytic choriomeningitis virus (LCMV) infection. A single dose of influenza virus [A/WSN/33 (H1N1)] bearing a cytotoxic T-lymphocyte-specific epitope of the LCMV nucleoprotein (residues 116 to 127) in the neuraminidase stalk protected mice against LCMV challenge for at least 4 months. The immunity was mediated by cytotoxic T lymphocytes and was haplotype specific, indicating that the observed protective response was solely a consequence of prior priming with the H-2d LCMV nucleoprotein epitope expressed in the recombinant influenza virus. We also found that as many as 58 amino acids could be inserted into the neuraminidase stalk without loss of viral function. These findings demonstrate the potential of influenza virus as a vaccine vector, with the neuraminidase stalk as a repository for foreign epitopes.

Amino Acid Sequence↗
PubMed1993

Prominent usage of V beta 8.3 T cells in the H-2Db-restricted response to an influenza A virus nucleoprotein epitope.

The spectrum of TCR usage has been analyzed for virus-specific CD8+ T cells isolated from the regional mediastinal lymph modes and from the lung by bronchoalveolar lavage (BAL) of C57BL/6 (B6) mice with influenza pneumonia. Lymphocytes were recovered during the acute phase of the primary response in mice infected with an H3N2 (A/HKx31) virus, or in immune animals that were secondarily challenged with an H1N1 virus (A/PR8). Cells taken directly from the BAL of infected mice exhibited an increase in the frequency of V beta 8.3+/CD8+ T cells. In addition, 20 to 50% of proliferating CD8+ T cells in the mediastinal lymph nodes and BAL populations stimulated in vitro with A/HKx31 were V beta 8.3 TCR+. These observations indicated that the V beta 8.3+/CD8+ T cells were specifically involved in the inflammatory process during influenza infection. However, in vivo depletion of V beta 8+ T cells in CD4-depleted mice did not adversely affect viral clearance, suggesting that other CD8+ T cells can compensate for the absence of these cells. The spectrum of TCR usage was also analyzed for influenza-specific T cell hybridomas derived from freshly isolated BAL of mice with pneumonia. Many of these T cell hybridomas were V beta 8.3+, although other TCR V beta elements were used. All of the V beta 8.3+ hybridomas recognized the H-2Db-restricted NP epitope, 365-380. Although the V beta 8.3 TCR contain similar TCR D beta and J beta elements, V alpha usage was surprisingly variable. Therefore, recognition of this particular epitope was dominated by the beta-chain of the TCR. We conclude that the murine CD8+ response to influenza A virus infection of B6 mice is limited in terms of the diversity of the responding T cells. However, there is significant plasticity in the CD8+ response, which readily compensates for the absence of the dominant T cell population.

Amino Acid Sequence↗
PubMed1993

Partitioning of responder CD8+ T cells in lymph node and lung of mice with Sendai virus pneumonia by LECAM-1 and CD45RB phenotype.

Patterns of LECAM-1 and CD45RB expression have been analysed for mediastinal lymph node (MLN) and bronchoalveolar lavage (BAL) populations from C57BL/6J mice with primary Sendai virus pneumonia. The findings indicate that virus-specific CD8+ CTL precursors differentiate in the regional lymph node and become effector CTL after localization to the virus-infected respiratory tract. Relatively few of the MLN CD8+ T cells were LECAM-1-, and all were CD45RB-hi throughout the acute and recovery phases of this disease process. The CD8+ CD45RB-hi and LECAM-1+ T cells characteristic of the MLN were more apparent in the BAL during the 1st wk after exposure to virus, with this shifting to a predominant CD8+ LECAM-1- CD45RB-lo phenotype from day 10 after infection. In contrast, the CD4+ set was generally CD45RB-lo LECAM-1- in the BAL, although CD4+ CD45RB-lo and LECAM-1- cells maintained at relatively high levels in the MLN. The virus-specific CD8+ effectors found in the BAL from day 8 after infection were uniformly LECAM-1-, but varied in the level of CD45RB expression. Evidence of CTL activity was minimal for the MLN, though virus-specific CTLp were present from day 5 after infection and reached maximum numbers within a further 2 days. The ratio of LECAM-1-:LECAM-1+ CTLp in the MLN ranged from 25:1 to > 200:1, with this pattern being maintained for memory spleen cells recovered 3 mo later. Lack of expression of LECAM-1 is thus characteristic of the great majority of Sendai-virus-specific CD8+ T cells, identified either as effector CTL or as precursors that can be stimulated in vitro under limiting dilution conditions. These lymphocyte populations cannot be discriminated on the basis of hi or lo CD45RB expression.

Animals↗
PubMed1993

Activation of cytokine genes in T cells during primary and secondary murine influenza pneumonia.

The patterns of cytokine mRNA expression in mice with primary or secondary influenza pneumonia have been assessed by in situ hybridization analysis of cells from both the mediastinal lymph node (MLN) and the virus-infected lung. Evidence of substantial transcriptional activity was found in all lymphocyte subsets recovered from both anatomical sites. The kinetics of cytokine mRNA expression after primary infection with an H3N2 virus were in accord with the idea that the initial response occurs in regional lymphoid tissue, with the effector T cells later moving to the lung. This temporal separation was much less apparent for the more rapid secondary response resulting from challenge of H3N2-primed mice with an H1N1 virus. Among the T cell receptor alpha/beta+ subsets, transcripts for interferon (IFN) gamma and tumor necrosis factor beta were most commonly found in the CD8+ population whereas mRNA for interleukin (IL) 4 and IL-10 was much more prevalent in CD4+ T cells. The gamma/delta T cells expressed mRNA for all cytokines tested, with IL-2, IL-4, and IFN-gamma predominating among those recovered from the inflammatory exudate. At particular time points, especially early in the MLN and late in the infected lung, the frequency of mRNA+ lymphocytes was much higher than would be expected from current understanding of the prevalence of virus-specific precursors and effectors. If this response is typical, induction of cytokine gene expression for T cells that are not responding directly to the invading pathogen may be a prominent feature of acute virus infections.

Animals↗
PubMed1993

hsp65 mRNA+ macrophages and gamma delta T cells in influenza virus-infected mice depleted of the CD4+ and CD8+ lymphocyte subsets.

The effects of depleting CD4+ and CD8+ T cells on macrophage recruitment have been analyzed for bronchoalveolar lavage (BAL) populations from mice with primary or secondary influenza pneumonia. Macrophages were characterized by both the capacity to engulf latex particles and the expression of mRNA for a 65 kD heat shock protein (hsp65). The localization of hsp65 mRNA+ cells to the pneumonic lung was greatly enhanced in the secondary response. Eliminating the CD4+ and CD8+ T cells decreased the prevalence of hsp65 mRNA+latex+ macrophages as much as seven-fold, though the frequency of latex+ cells was higher in the residual inflammatory process. The CD4-8- gamma delta T cells were also relatively enriched in the BAL from the depleted mice. However, the localization of gamma delta T cells to the pneumonic lung does not compensate either quantitatively or qualitatively for the lack of the CD4+ and CD8+ alpha beta T-cell subsets, which are responsible for activating a substantial proportion of the phagocytic cells to express transcripts of an endogenous hsp65 gene.

Animals↗
PubMed1993

Lymphocytic choriomeningitis virus induces a chronic wasting disease in mice lacking class I major histocompatibility complex glycoproteins.

Lymphocytic choriomeningitis virus (LCMV) induces a chronic, wasting syndrome when injected intracerebrally into H-2b mice homozygous for a beta 2-microglobulin (beta 2-m (-/-)) gene disruption. These mice have very few CD8+ T cells and express little class I MHC glycoprotein, though minimal levels of the H-2Db molecule have been detected on in vitro cultured beta 2-m (-/-) cells. The underlying immunopathological process in these beta 2-m (-/-) mice is mediated by virus immune CD4+ effectors. However, adoptively transferred CD8+ T cells from normal, LCMV-infected H-2Db compatible donors induce significant (but low level) meningitis in beta 2-m (-/-) recipients. Such mice develop neither the neurological disease characteristic of LCM nor the persistent, though generally non-fatal, debility that occurs when only the CD4+ T cell subset is involved.

Animals↗
PubMed1993

Virus infections in mice with targeted gene disruptions.

The experimental dissection of the ways that the various cells and molecules of the immune system interact to promote virus clearance has been greatly facilitated by the availability of mice with targeted disruptions of key genes. New insights are emerging, and details of host resistance mechanisms that could only be inferred for the in vivo situation are now being clearly established.

Animals↗
PubMed1993

Divergence between cytotoxic effector function and tumor necrosis factor alpha production for inflammatory CD4+ T cells from mice with Sendai virus pneumonia.

Sendai virus pneumonia in beta 2-microglobulin-deficient [beta 2-m(-/-)] mice lacking CD8+ T cells is characterized by the development of CD4+ cytotoxic T lymphocytes that can be recovered directly from the respiratory tract. These CD4+ cytotoxic T lymphocytes are not found in beta 2-m (+/+) mice, though inflammatory CD4+ T cells from both beta 2-m (-/-) and beta 2-m (+/+) mice produce substantial amounts of tumor necrosis factor alpha. Blocking experiments with a monoclonal antibody that also inhibits tumor necrosis factor beta show that the secreted forms of these two cytokines are not responsible for virus-specific killing of class II major histocompatibility complex-compatible targets. Comparison of electron micrographs indicates that the CD4+ effectors from the beta 2-m (-/-) mice are potent inducers of apoptosis, while this is not the case for the beta 2-m (+/+) CD4+ set. These experiments further define the functional status of virus-specific CD4+ T cells responding in vivo in the presence or absence of CD8+ effectors.

Animals↗
PubMed1992

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.

Animals↗
PubMed1992

Analyzing the distribution of cells expressing mRNA for T cell receptor gamma and delta chains in a virus-induced inflammatory process.

Acute inflammatory processes are extremely complex, containing sets of activated cells that may be difficult to categorize. The interface between two methodologies for characterizing the involvement of gamma delta T cells, in situ hybridization to detect T cell receptor (TCR) mRNA and flow cytometric analysis of surface TCR expression, is utilized here to study the pneumonia caused by intranasal (i.n.) infection of mice with influenza A viruses. Substantial numbers of cells expressing mRNA for the gamma and delta TCR chains are present in bronchoalveolar lavage (BAL) populations obtained either late in the course of primary infection with an H3N2 virus or within a few days of secondary challenge with an H1N1 virus. The majority of the gamma delta TCR mRNA+ cells detected in FACS-separated BAL populations partition to the Thy1+ gamma delta TCR+ subset, while relatively few (less than 10%) C delta mRNA transcripts are found in cells that phagocytose latex particles. However, an additional set of gamma delta TCR mRNA+ cells is also located in a high side scatter (H-SSC) population, which stains nonspecifically with monoclonal antibodies (mAbs) and is normally gated out in the process of flow cytometric analysis. This H-SSC population tends to be enriched for cells expressing C gamma 1/2 rather than C gamma 4 mRNA. While some gamma delta TCR+ lymphocytes can be demonstrated by in vitro stimulation of the CD3 epsilon+ subset within this H-SSC population, the majority of the gamma delta T cell precursors that can be expanded in culture demonstrate a low side scatter (L-SSC) profile more characteristic of normal T lymphocytes. The possibility that subsets of activated, granular (H-SSC) alpha beta TCR+ and C gamma 1/2 mRNA+ gamma delta T cells are being missed when conventional FACS analysis is used to study this viral pneumonia is discussed.

Animals↗
PubMed1992

Extent of gamma delta T cell involvement in the pneumonia caused by Sendai virus.

The prevalence of gamma delta T cells in bronchoalveolar lavage (BAL) populations recovered from the respiratory tract of young, adult C57BL/6J mice infected intranasally (i.n.) with Sendai virus has been assessed by FACS-phenotyping, and by probing cytocentrifuge preparations for expression of TCR gamma mRNA. The surface gamma delta TCR+ set comprised from 5 to 20% of the inflammatory lymphocytes in sequential samples taken throughout the course of this nonfatal viral pneumonia. The BAL population also contained numerous cells expressing mRNA for C gamma 1/2 and C gamma 4; the C-regions were utilized for productive TCR gene rearrangement. Sorting the lymphocytes from the BAL established that greater than 90% of both the TCR gamma and TCR beta mRNA partitioned to cells with the appropriate surface TCR phenotype, while less than 7% of the TCR mRNA+ cells in the total inflammatory exudate were phagocytes that engulfed latex particles. Both the frequency and the total numbers of the gamma delta TCR+ and TCR gamma mRNA+ cells were increased in mice depleted of alpha beta T cells by in vivo treatment with mAbs to CD4 and CD8, indicating that the CD4+ and CD8+ alpha beta and CD4-8- gamma delta T cell subsets may operate independently in this virus disease. The C gamma 1/2 mRNA phenotype predominated throughout the course of the active infection, with a transition to maximal prevalence of the C gamma 4 mRNA+ set occurring very late (Day 20) in the resolving inflammatory process. Large numbers of macrophages expressing mRNA (greater than 50%) for a mammalian 65-kDa heat shock protein (hsp65), a possible target for some of the gamma delta T cells, were present early (Days 5-7) and remained at lower levels (less than 20%) thereafter. These hsp65 mRNA+ macrophages were much less apparent in BAL populations from mice depleted concurrently of the CD4+ and CD8+ T cell subsets, indicating that exposure to Sendai virus alone is not the major factor inducing the transcription of this endogenous gene. These experiments thus establish that gamma delta T cells are a minority of the infiltrating lymphocytes in Sendai virus pneumonia and provide new insights into the spectrum of hsp65 mRNA and TCR gamma mRNA expression during an inflammatory process.

Animals↗
PubMed1992

Roles of alpha beta and gamma delta T cell subsets in viral immunity.

The current status of T-cell subset involvement in viral immunity is summarized for experimental studies in mice. The immunobiology of the normal host response is discussed, with particular reference to lymphocytic choriomeningitis (LCM) and influenza. The general impression is that CD8+ cytotoxic T lymphocytes, CD4+ TH1 cells, gamma interferon, and IL-2 are of major importance, with these different components of the immune system interacting to promote an optimal response. However, experiments with a variety of virus systems indicate that there is considerable plasticity, at least in young, adult mice. Other mechanisms often compensate if a key lymphocyte subset is absent throughout the development of the immune response. Influenza-infected mice depleted of either CD4+ or CD8+ T cells clear virus and recover, though the latter may not be true for the elimination of LCM virus. Emerging information on the involvement of gamma delta T cells in viral pneumonia is summarized, but there is as yet no understanding of the biological significance (if any) of these lymphocytes in viral immunity. The point is made that alpha beta T-cell memory to viruses is long-lived, and the need for antigen persistence to maintain such memory is questioned.

Animals↗
PubMed1991

Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells.

Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.

Animals↗
PubMed1991

Activation status of the CD4-8- gamma delta-T cells recovered from mice with influenza pneumonia.

The role that gamma delta-T lymphocytes play in virus infections is yet to be defined. The TCR-gamma delta + cell population found late in the course of influenza pneumonia has been analyzed for ligand-dependent lytic function. These gamma delta-T cells are not constitutively cytotoxic when recovered directly from the site of virus-induced damage in the respiratory tract, although the TCR-alpha beta + population that is present concurrently contains such lytic effectors. Both sets of lymphocytes mediate cytotoxic activity after further in vitro stimulation in the presence of mAb to CD3 and low concentrations of rIL-2. Secondary stimulation in vivo with a cross-reactive influenza A virus does not lead to the emergence of a cytotoxic gamma delta-T cell population, although substantial numbers of these gamma delta-T cells express mRNA for a variety of lymphokines and cytokines. Analysis of DNA content indicates that many of the gamma delta-T cells isolated directly from the pneumonic lung are cycling. This could reflect continuing stimulation by a specific ligand, perhaps a self-component expressed at abnormally high levels in the site of virus-induced pathology. However, we could find no evidence to indicate that the gamma delta-T cells are acting to eliminate redundant components of the host response. The percentage of inflammatory macrophages and nonphagocytic cells expressing mRNA for a 65-kDa heat-shock protein (the proposed target for at least a subset of these gamma delta-T cells) is not reduced during the time that lymphocytes with mRNA for the TCR-gamma delta are present in greatest numbers. Possible alternative functions for the gamma delta-T cells are discussed.

Animals↗