Biomedical subjects

J D Altman

Publications and source records attributed to J D Altman.

At least 37 records · Page 2Linked to original sources

PubMed2000

Peptide-MHC class I tetrameric complexes display exquisite ligand specificity.

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8(+) T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.

Antigen Presentation↗

PubMed2000

Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections are characterized by early peaks of viraemia that decline as strong cellular immune responses develop. Although it has been shown that virus-specific CD8-positive cytotoxic T lymphocytes (CTLs) exert selective pressure during HIV and SIV infection, the data have been controversial. Here we show that Tat-specific CD8-positive T-lymphocyte responses select for new viral escape variants during the acute phase of infection. We sequenced the entire virus immediately after the acute phase, and found that amino-acid replacements accumulated primarily in Tat CTL epitopes. This implies that Tat-specific CTLs may be significantly involved in controlling wild-type virus replication, and suggests that responses against viral proteins that are expressed early during the viral life cycle might be attractive targets for HIV vaccine development.

AIDS Vaccines↗

PubMed2000

Effect of Staphylococcus enterotoxin B on the concurrent CD8(+) T cell response to influenza virus infection.

Bacterial superantigens have potent in vivo effects. Respiratory viral infections are often associated with secondary bacterial infections, raising the likelihood of exposure to bacterial superantigens after the initiation of the anti-viral immune response. In this study, the general and V beta-specific effects of exposure to Staphylococcal enterotoxin B (SEB) during influenza virus infection on both the ongoing acute and the subsequent recall CD8(+) T cell responses were analyzed, using the well-characterized murine influenza model system and tetrameric MHC/peptide reagents to directly identify virus-specific T cells. The results show that although superantigen exposure during the primary viral infection caused delayed viral clearance, there was remarkably little effect of SEB on the magnitude or TCR repertoire of the ongoing cytolytic T cell response or on the recall response elicited by secondary viral infection. Thus, despite the well-characterized immunomodulatory effects of SEB, there was surprisingly little interference with concurrent anti-viral immunity.

Acute Disease↗

PubMed2000

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Adjuvants, Immunologic↗

PubMed2000

Cutting edge: increased expression of Bcl-2 in antigen-specific memory CD8+ T cells.

Bcl-2 plays a critical role in regulating cell survival and apoptosis. We examined Bcl-2 expression in virus-specific CD8 T cells during the expansion, death, and memory phases of the T cell response following infection of mice with lymphocytic choriomeningitis virus (LCMV). Naive CD8 T cells expressed a basal level of Bcl-2 that was down-regulated in effector CD8 T cells just before the death phase. Bcl-2 levels remained low during the death phase but surviving memory CD8 T cells expressed higher levels of Bcl-2 than naive cells. These changes were shown to occur in LCMV TCR transgenic cells as well as virus-specific CD8 T cells in C57BL/6 and BALB/c mice identified by MHC class I tetramers. In all instances, memory CD8 T cells expressed higher levels of Bcl-2, suggesting that increased Bcl-2 expression plays a role in the long-term maintenance of memory CD8 T cells in vivo.

Animals↗

PubMed2000

Differentiating between memory and effector CD8 T cells by altered expression of cell surface O-glycans.

Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.

Animals↗

PubMed2000

Postexposure vaccination massively increases the prevalence of gamma-herpesvirus-specific CD8+ T cells but confers minimal survival advantage on CD4-deficient mice.

Mice that lack CD4(+) T cells remain clinically normal for more than 60 days after respiratory challenge with the murine gamma-herpesvirus 68 (gammaHV-68), then develop symptoms of a progressive wasting disease. The gammaHV-68-specific CD8(+) T cells that persist in these I-A(b-/-) mice are unable to prevent continued, but relatively low level, virus replication. Postexposure challenge with recombinant vaccinia viruses expressing gammaHV-68 lytic cycle epitopes massively increased the magnitude of the gammaHV-68-specific CD8(+) population detectable by staining with tetrameric complexes of MHC class I glycoprotein + peptide, or by interferon-gamma production subsequent to in vitro restimulation with peptide. The boosting effect was comparable for gammaHV-68-infected I-A(b-/-) and I-A(b+/+) mice within 7 days of challenge, and took more than 110 days to return to prevaccination levels in the I-A(b+/+) controls. Although the life-span of the I-A(b-/-) mice was significantly increased, there was no effect on long-term survival. A further boost with a recombinant influenza A virus failed to improve the situation. Onset of weight loss was associated with a decline in gammaHV-68-specific CD8(+) T cell numbers, though it is not clear whether this was a cause or an effect of the underlying pathology. Even very high levels of virus-specific CD8(+) T cells thus provide only transient protection against the uniformly lethal consequences of gammaHV-68 infection under conditions of CD4(+) T cell deficiency.

Animals↗

PubMed2000

4-1BB costimulation is required for protective anti-viral immunity after peptide vaccination.

Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (-/-) and wild-type (+/+) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396-404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-gamma staining, and IFN-gamma enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBL-/- mice were augmented to levels similar to those in 4-1BBL+/+ mice. Two months after immunization, 4-1BBL+/+ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBL-/- mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination.

Animals↗

PubMed2000

Soluble antigen and CD40 triggering are sufficient to induce primary and memory cytotoxic T cells.

The signals directing induction of tolerance rather than immunity are largely unknown. The CD8 T cell response to soluble Ags generally results in deletional tolerance following transient, costimulation-dependent activation. We demonstrated that CD40 signaling reversed the outcome of this response. Adoptive transfer of OVA-specific CD8 T cells followed by soluble OVA immunization resulted in induction of lytic activity and optimal clonal expansion only when CD40 was triggered via an agonistic mAb. Activation of CD8 T cells by CD40 signaling was indirect, because CD40 expression by host cells was required. CD40 signaling along with soluble Ag immunization also induced expansion of secondary lymphoid and intestinal mucosal endogenous OVA-specific CD8 T cells as detected by MHC tetramer reactivity. When CD40 activation was included, long-lived secondary lymphoid and mucosal memory CD8 cells were generated from adoptively transferred and endogenous CD8 T cells. Mucosal and peripheral CD8 memory cells exhibited constitutive Ag-specific lytic activity, with mucosal memory cells being 10-fold more lytic than splenic or lymph node memory cells. These results demonstrated that CD40 signaling during a response to a poorly immunogenic soluble Ag was necessary and sufficient for CTL and memory T cell induction.

Adoptive Transfer↗

PubMed2000

Role of CD8beta domains in CD8 coreceptor function: importance for MHC I binding, signaling, and positive selection of CD8+ T cells in the thymus.

The contribution of the CD8beta subunit to CD8 coreceptor function is poorly understood. We now demonstrate that the CD8beta extracellular domain increases the avidity of CD8 binding to MHC I, and that the intracellular domain of CD8beta enhances association with two intracellular molecules required for TCR signal transduction, Lck and LAT. By assessing CD8+ T cell differentiation in CD8beta-deficient mice reconstituted with various transgenic CD8beta chimeric molecules, we also demonstrate that the intracellular and extracellular domains of CD8beta can contribute independently to CD8+ T cell development, but that both CD8beta domains together are most efficient. Thus, this study identifies the molecular functions of the CD8beta intracellular and extracellular domains and documents their contributions to CD8+ T cell development.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Viremia control following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques.

Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

Animals↗

PubMed2000

Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus.

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

Animals↗

PubMed2000

A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8(+) T-cell response is much less apparent following secondary challenge.

Respiratory challenge of H-2(b) mice with an H3N2 influenza A virus causes an acute, transient pneumonitis characterized by the massive infiltration of CD8(+) T lymphocytes. The inflammatory process monitored by quantitative analysis of lymphocyte populations recovered by bronchoalveolar lavage is greatly enhanced by prior exposure to an H1N1 virus, with the recall of cross-reactive CD8(+)-T-cell memory leading to more rapid clearance of the infection from the lungs. The predominant epitope recognized by the influenza virus-specific CD8(+) set has long been thought to be a nucleoprotein (NP(366-374)) presented by H-2D(b) (D(b)NP(366)). This continues to be true for the secondary H3N2-->H1N1 challenge but can no longer be considered the case for the primary response to either virus. Quantitative analysis based on intracellular staining for gamma interferon has shown that the polymerase 2 protein (PA(224-233)) provides a previously undetected epitope (D(b)PA(224)) that is at least as prominent as D(b)NP(366) during the first 10 days following primary exposure to either the H3N2 or H1N1 virus. The response to D(b)NP(366) seems to continue for longer, even when infectious virus can no longer be detected, but there is no obvious difference in the prevalence of memory T cells specific for D(b)NP(366) and D(b)PA(224). The generalization that the magnitude of the functional memory T-cell pool is a direct consequence of the clonal burst size during the primary response may no longer be useful. Previous CD8(+)-T-cell immunodominance heirarchies defined largely by cytotoxic T-lymphocyte assays may need to be revised.

Amino Acid Sequence↗

PubMed2000

Polyomavirus-infected dendritic cells induce antiviral CD8(+) T lymphocytes.

CD8(+) T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8(+) T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8(+) T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8(+) T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8(+) T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8(+) T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8(+) T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8(+) T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8(+) T cell surveillance against polyomavirus-induced tumors.

3T3 Cells↗

PubMed1999

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.

Animals↗

PubMed1999

Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission.

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.

Adrenergic alpha-Agonists↗

PubMed1999

Visualization of polyoma virus-specific CD8+ T cells in vivo during infection and tumor rejection.

T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389-397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389-397 peptide to directly visualize and enumerate MT389-397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute approximately 20% of the total and approximately 40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389-397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-gamma after 6-h in vitro stimulation by MT389-397 peptide. Consistent with preferential Vbeta6 expression by MT389-397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vbeta gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389-397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.

Acute Disease↗

PubMed1999

Inverted immunodominance and impaired cytolytic function of CD8+ T cells during viral persistence in the central nervous system.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) clear infectious virus; nevertheless, virus persists in the CNS as noninfectious RNA, resulting in ongoing primary demyelination. Phenotypic and functional analysis of CNS infiltrating cells during acute infection revealed a potent regional CD8+ T cell response comprising up to 50% virus-specific T cells. The high prevalence of virus-specific T cells correlated with ex vivo cytolytic activity and efficient reduction in viral titers. Progressive viral clearance coincided with the loss of cytolytic activity, but retention of IFN-gamma secretion and increased expression of the early activation marker CD69, indicating differential regulation of effector function. Although the total number of infiltrating T cells declined following clearance of infectious virus, CD8+ T cells, both specific for the dominant viral epitopes and of unknown specificity, were retained within the CNS, suggesting an ongoing T cell response during persistent CNS infection involving a virus-independent component. Reversed immunodominance within the virus-specific CD8+ T cell population further indicated epitope-specific regulation, supporting ongoing T cell activation. Even in the absence of infectious virus, the CNS thus provides an environment that maintains both unspecific and Ag-specific CD8+ T cells with restricted effector function. Chronic T cell stimulation may thus play a role in preventing viral recrudescence, while increasing the risk of pathological conditions, such as demyelination.

Acute Disease↗