Biomedical subjects

John D Altman

Publications and source records attributed to John D Altman.

32 records · Page 2Linked to original sources

PubMed2002

CD94/NKG2 expression does not inhibit cytotoxic function of lymphocytic choriomeningitis virus-specific CD8+ T cells.

Murine Ag-specific CD8(+) T cells express various NK markers and NK inhibitory receptors that have been proposed to modulate immune responses. Following acute infection of C57BL/6 and BALB/cJ mice with lymphocytic choriomeningitis virus (LCMV), we observed that Ag-specific CD8(+) T cells expressed CD94/NKG2. Only slight expression of Ly49A and Ly49C receptors was observed on NP396-specific T cells, while all NP396-specific T cells expressed the NKT cell marker U5A2-13 Ag. Expression of CD94/NKG2 was maintained for at least 1 year following LCMV infection, as was the NKT cell marker. By means of cell sorting and quantitative PCR, we found that NP118-specific CD8(+) T cells primarily express transcripts for inhibitory NKG2 receptor isoforms. CD94/NKG2 expression was also observed on Ag-specific CD8(+) T cells following infection with polyoma virus, influenza virus, and Listeria monocytogenes, suggesting that it may be a common characteristic of Ag-specific CD8(+) T cells following infection with viral or bacterial pathogens. Expression of CD94/NKG2 on memory-specific CD8(+) T cells did not change following secondary challenge with LCMV clone 13 and did not inhibit viral clearance. Furthermore, we found no evidence that CD94/NKG2 inhibits either the lytic function of LCMV-specific T cells or their capacity to produce effector cytokines upon peptide stimulation. Finally, down-regulation of CD94/NKG2 was found to occur only during chronic LCMV infection. Altogether, this study suggests that CD94/NKG2 expression is not necessarily correlated with inhibition of T cell function.

Animals↗

PubMed2002

In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses.

Given the key role CD8+ T cells play in controlling viral infection, strategies to enhance these responses may have important clinical applications. We found that in vivo CD137 stimulation with an agonistic monoclonal antibody enhanced the primary CD8+ T cell response to influenza type A viral infection in mice. Stimulation of CD137 increased the absolute number of CD8+ T cells to influenza epitopes in the lungs of infected animals, preferentially expanded CD8+ T cells that recognized nondominant epitopes and greatly enhanced direct ex vivo cytotoxicity. CD137 stimulation also restored the CD8+ T cell response to the immunodominant influenza epitope in CD28-/- mice. Thus, in vivo CD137 stimulation enhances and broadens the CD8+ T cell response to influenza virus and can restore the CD8+ T cell response when CD28 costimulation is absent. This suggests that CD137 stimulation may be useful as a strategy to enhance the CD8+ T cell response to viruses.

Animals↗

PubMed2002

Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine.

Heterologous prime/boost regimens have the potential for raising high levels of immune responses. Here, we report that DNA priming followed by a recombinant modified vaccinia Ankara (rMVA) booster has controlled a highly pathogenic immunodeficiency virus challenge in a Rhesus macaque model. Both the DNA and rMVA components of the vaccine expressed multiple immunodeficiency virus proteins. Two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster. These highly promising findings provide hope that a relatively simple multiprotein DNA/MVA vaccine can help to control the AIDS epidemic.

AIDS Vaccines↗

PubMed2002

Estimating the precursor frequency of naive antigen-specific CD8 T cells.

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner. The precursor frequency is equal to the number of transferred cells when the transgenic and endogenous responses are of equal magnitude. Using this method we have estimated the precursor frequency of naive CD8 T cells specific for the H-2D(b)-restricted GP33-41 epitope of LCMV to be 1 in 2 x 10(5). Thus, in an uninfected mouse containing approximately 2-4 x 10(7) naive CD8 T cells we estimate there to be 100-200 epitope-specific cells. After LCMV infection these 100-200 GP33-specific naive CD8 T cells divide >14 times in 1 wk to reach a total of approximately 10(7) cells. Approximately 5% of these activated GP33-specific effector CD8 T cells survive to generate a memory pool consisting of approximately 5 x 10(5) cells. Thus, an acute LCMV infection results in a >1,000-fold increase in precursor frequency of D(b)GP33-specific CD8 T cells from 2 x 10(2) naive cells in uninfected mice to 5 x 10(5) memory cells in immunized mice.

Adoptive Transfer↗

PubMed2002

Visualization and quantification of T cell-mediated cytotoxicity using cell-permeable fluorogenic caspase substrates.

We have developed a non-radioactive flow-cytometry assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This flow-cytometry CTL (FCC) assay is predicated on measurement of CTL-induced caspase activation in target cells through detection of the specific cleavage of fluorogenic caspase substrates. Here we show that this assay reliably detects antigen-specific CTL killing of target cells, and demonstrate that it provides a more sensitive, more informative and safer alternative to the standard 51Cr-release assay most often used to quantify CTL responses. The FCC assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the FCC assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

Animals↗

PubMed2002

Induction of anti-simian immunodeficiency virus cellular and humoral immune responses in rhesus macaques by peptide immunogens: correlation of CTL activity and reduction of cell-associated but not plasma virus load following challenge.

Lipopeptides which carry the N-terminal moiety tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl (P(3)CSS) have been shown to have effective adjuvant and transmembrane carrier properties. To test the ability of these constructs to immunize against simian immunodeficiency virus [(SIV)(mac)] infection, rhesus macaques, prescreened for expression of the Mamu-A*01 MHC class I molecule, were immunized at regular intervals with lipopeptides corresponding to known SIV(mac) CTL epitopes alone or in combination with multiple antigenic peptides corresponding to neutralizing epitopes. Both humoral and CTL responses were elicited and the monkeys, along with non-immunized control animals, were challenged intravenously with 20 MID(50) of the homologous, uncloned SIV(mac251-32H) grown in rhesus monkey PBMC. Although none of the monkeys were protected from infection, most demonstrated an anamnestic CTL response with epitope-specific CTL precursor frequencies reaching as high as 1 in 20 total PBMC as measured by limiting dilution CTL assay or 25% of all CD8(+) T-cells using tetrameric MHC-I/peptide complexes. A significant inverse correlation between the levels of CTLp and the number of infected cells in circulation was observed. However, no such correlation with the plasma viral load (RNA copies/ml) was evident.

Amino Acid Sequence↗

PubMed2002

Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine.

Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4(+) cells. Interestingly, most of the CD4(+) cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4(+) cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4(+) cells.

AIDS Vaccines↗

PubMed2002

Bacterial superantigen exposure after resolution of influenza virus infection perturbs the virus-specific memory CD8(+)-T-cell repertoire.

Heterologous viral infections have been shown to impact the preexisting memory CD8(+)-T-cell repertoire. Bacterial superantigens are products of common human pathogenic bacteria, including staphylococci and streptococci, that are potent T-cell-stimulatory molecules. In this report, we show that exposure to staphylococcal enterotoxin B, a bacterial superantigen, causes a selective functional deletion of cross-reactive influenza virus-specific CD8(+) memory T cells. This perturbation of the memory repertoire can have a significant impact on viral clearance after secondary challenge.

Animals↗

PubMed2002

Different patterns of immune responses but similar control of a simian-human immunodeficiency virus 89.6P mucosal challenge by modified vaccinia virus Ankara (MVA) and DNA/MVA vaccines.

Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.

AIDS Vaccines↗

PubMed2002

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.

Acute Disease↗

PubMed2002

Effects of cytotoxic T lymphocytes (CTL) directed against a single simian immunodeficiency virus (SIV) Gag CTL epitope on the course of SIVmac239 infection.

Vaccine-induced cytotoxic T lymphocytes (CTL) have been implicated in the control of virus replication in simian immunodeficiency virus (SIV)-challenged and simian-human immunodeficiency virus-challenged macaques. Therefore, we wanted to test the impact that vaccine-induced CTL responses against an immunodominant Gag epitope might have in the absence of other immune responses. By themselves, these strong CTL responses failed to control SIVmac239 replication.

Animals↗

PubMed2002

Role of lymphotoxin alpha in T-cell responses during an acute viral infection.

The importance of lymphotoxin alpha (LTalpha) in lymphoid organogenesis is well established. Although LTalpha has been implicated in the pathogenesis of T-cell-mediated immunopathologies, the requirement for LTalpha in T-cell activation and effector function in vivo is not well understood. To determine the role of LTalpha in T-cell activation in vivo, we compared the generation of antigen-specific T-cell responses between wild type (+/+) and LTalpha-deficient (LTalpha(-/-)) mice during an acute infection with lymphocytic choriomeningitis virus (LCMV). Our studies showed that LCMV-infected LTalpha(-/-) mice had a profound impairment in the activation and expansion of virus-specific CD8 T cells in the spleen, as determined by cytotoxicity assays, intracellular staining for gamma interferon, and staining with major histocompatibility complex class I tetramers. Further, the nonlymphoid organs of LTalpha(-/-) mice also contained substantially lower number of LCMV-specific CD8 T cells than those of +/+ mice. Greatly reduced virus-specific CD8 T-cell responses in LTalpha(-/-) mice led to a defect in LCMV clearance from the tissues. In comparison to that in +/+ mice, the activation of LCMV-specific CD4 T cells was also significantly attenuated in LTalpha(-/-) mice. Adoptive transfer experiments were conducted to determine if abnormal lymphoid architecture in LTalpha(-/-) mice caused the impairment in the activation of LCMV-specific T-cell responses. Upon adoptive transfer into +/+ mice, the activation and expansion of LCMV-specific LTalpha(-/-) T cells were restored to levels comparable to those of +/+ T cells. In a reciprocal cell transfer experiment, activation of +/+ T cells was significantly reduced upon transfer into LTalpha(-/-) mice. These results showed that impairment in the activation of LCMV-specific T cells in LTalpha(-/-) mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LTalpha(-/-) T cells.

Adoptive Transfer↗

PubMed2002

Tat-vaccinated macaques do not control simian immunodeficiency virus SIVmac239 replication.

The regulatory proteins of human immunodeficiency virus may represent important vaccine targets. Here we assessed the role of Tat-specific cytotoxic T lymphocytes (CTL) in controlling pathogenic simian immunodeficiency virus SIVmac239 replication after using a DNA-prime, vaccinia virus Ankara-boost vaccine regimen. Despite the induction of Tat-specific CTL, there was no significant reduction in either peak or viral set point compared to that of controls.

AIDS Vaccines↗

PubMed2002

Major histocompatibility complex (MHC) tetramer technology: an evaluation.

Single-cell assays are currently favored to quantitate T-cell responses. Staining antigen-specific T-cells with fluorescently labeled tetrameric major histocompatibility complex (MHC)/peptide complexes has greatly enhanced the ability to assess the cellular dynamics of an immune response at the single-cell level. This article reviews MHC tetramer technology, defining it, discussing how MHC tetramers are made, outlining the benefits of this technology, comparing and contrasting it to other methods for evaluating immune responses, and describing current applications.

Bias↗