Biomedical subjects

J D Altman

Publications and source records attributed to J D Altman.

At least 73 records · Page 4Linked to original sources

PubMed1998

Characteristics of virus-specific CD8(+) T cells in the liver during the control and resolution phases of influenza pneumonia.

Dissection of the primary and secondary response to an influenza A virus established that the liver contains a substantial population of CD8(+) T cells specific for the immunodominant epitope formed by H-2Db and the influenza virus nucleoprotein peptide fragment NP366-374 (DbNP366). The numbers of CD8(+) DbNP366(+) cells in the liver reflected the magnitude of the inflammatory process in the pneumonic lung, though replication of this influenza virus is limited to the respiratory tract. Analysis of surface phenotypes indicated that the liver CD8(+) DbNP366(+) cells tended to be more "activated" than the set recovered from lymphoid tissue but generally less so than those from the lung. The distinguishing characteristic of the lymphocytes from the liver was that the prevalence of the CD8(+) DbNP366(+) set was always much higher than the percentage of CD8(+) T cells that could be induced to synthesize interferon gamma after short-term, in vitro stimulation with the NP366-374 peptide, whereas these values were generally comparable for virus-specific CD8(+) T cells recovered from other tissue sites. Also, the numbers of apoptotic CD8(+) T cells were higher in the liver. The results overall are consistent with the idea that antigen-specific CD8(+) T cells are destroyed in the liver during the control and resolution phases of this viral infection, though this destruction is not necessarily an immediate process.

Animals↗

PubMed1998

Conserved T cell receptor repertoire in primary and memory CD8 T cell responses to an acute viral infection.

Viral infections often induce potent CD8 T cell responses that play a key role in antiviral immunity. After viral clearance, the vast majority of the expanded CD8 T cells undergo apoptosis, leaving behind a stable number of memory cells. The relationship between the CD8 T cells that clear the acute viral infection and the long-lived CD8 memory pool remaining in the individual is not fully understood. To address this issue, we examined the T cell receptor (TCR) repertoire of virus-specific CD8 T cells in the mouse model of infection with lymphocytic choriomeningitis virus (LCMV) using three approaches: (a) in vivo quantitative TCR beta chain V segment and complementarity determining region 3 (CDR3) length repertoire analysis by spectratyping (immunoscope); (b) identification of LCMV-specific CD8 T cells with MHC class I tetramers containing viral peptide and costaining with TCR Vbeta-specific antibodies; and (c) functional TCR fingerprinting based on recognition of variant peptides. We compared the repertoire of CD8 T cells responding to acute primary and secondary LCMV infections, together with that of virus-specific memory T cells in immune mice. Our analysis showed that CD8 T cells from several Vbeta families participated in the anti-LCMV response directed to the dominant cytotoxic T lymphocyte (CTL) epitope (NP118-126). However, the bulk (approximately 70%) of this CTL response was due to three privileged T cell populations systematically expanding during LCMV infection. Approximately 30% of the response consisted of Vbeta10+ CD8 T cells with a beta chain CDR3 length of nine amino acids, and 40% consisted of Vbeta8.1+ (beta CDR3 = eight amino acids) and Vbeta8.2+ cells (beta CDR3 = six amino acids). Finally, we showed that the TCR repertoire of the primary antiviral CD8 T cell response was similar both structurally and functionally to that of the memory pool and the secondary CD8 T cell effectors. These results suggest a stochastic selection of memory cells from the pool of CD8 T cells activated during primary infection.

Animals↗

PubMed1998

In vivo dynamics of anti-viral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection.

Viral infections induce extensive T cell proliferation in vivo. However, only a small fraction (1-5%) of the activated T cells have been shown to be virus specific leading to the prevailing notion that most of the T cell expansion represents cytokine-mediated by-stander activation and/or cross reactive stimulation of non specific cells. To re-examine this issue we quantitated antigen specific CD8 T cells during acute LCMV infection of mice using three sensitive techniques: (i) intracellular cytokine production, (ii) single cell ELISPOT and (iii) direct visualization of antigen specific CD8 T cells by staining with MHC class I tetramers + peptide. In contrast to previous estimates, we found that 50-70% of the activated CD8 T cells were LCMV specific. This represented > or = 10,000-fold increase (approximately 2 x 10(7) virus specific cells/spleen) in 8 days with the peak expansion occurring between day 3 and 5 during which period virus specific CD8 T cells had an estimated division time of approximately 8 hours. Following viral clearance, the number of antigen specific CD8 T cells dropped to 1 x 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, memory CD8 T cells rapidly proliferated and again comprised > 50% of the total CD8 T cells. In contrast, upon challenge with a heterologous virus such as vaccinia, there was no change in the number of LCMV specific memory CTL, despite a substantial increase in the number of activated CD8 T cells. Taken together, these results show that much of the CD8 T cell expansion seen during viral infection represents antigen specific cells.

Animals↗

PubMed1998

Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.

Viral infections induce extensive T cell proliferation in vivo, but the specificity of the majority of the responding T cells has not been defined. To address this issue we used tetramers of MHC class I molecules containing viral peptides to directly visualize antigen-specific CD8 T cells during acute LCMV infection of mice. Based on tetramer binding and two sensitive assays measuring interferon-gamma production at the single-cell level, we found that 50%-70% of the activated CD8 T cells were LCMV specific [2 x 10(7) virus-specific cells/spleen]. Following viral clearance, antigen-specific CD8 T cell numbers dropped to 10(6) per spleen and were maintained at this level for the life of the mouse. Upon rechallenge with LCMV, there was rapid expansion of memory T cells, but after infection with the heterologous vaccinia virus there was no detectable change in the numbers of LCMV-specific memory CTL. Therefore, much of the CD8 T cell expansion seen during viral infection represents antigen-specific cells and warrants a revision of our current thinking on the size of the antiviral response.

Animals↗

PubMed1998

Induction of rapid T cell activation and tolerance by systemic presentation of an orally administered antigen.

To understand how orally introduced antigen regulates peripheral immune responses, we fed cytochrome c protein to mice transgenic for the beta chain of a cytochrome c-specific TCR and followed the antigen-specific T cell responses with a cyt c/I-Ek tetramer staining reagent. We find that within 6 hr of cytochrome c administration, antigen-specific systemic T cell activation is induced, and spleen cells gain the ability to stimulate cytochrome c-specific T cell responses. Feeding multiple low doses of cytochrome c down-regulates the systemic immune response, which can be correlated with a reduction of antigen-specific T cells and not with immune deviation. These results suggest that systemic distribution of antigen contributes significantly to oral tolerance induction.

Administration, Oral↗

PubMed1998

Virus-specific CD8+ T cells in primary and secondary influenza pneumonia.

Virus-specific CD8+ effector T cells (eCTL) are enriched in the lungs of mice with primary influenza pneumonia, though later detection of memory T cells (mCTL) in the mediastinal lymph nodes (MLN) or spleen by peptide-based staining protocols is at the limits of flow cytometric analysis. Respiratory challenge with an H3N2 virus months after H1N1 priming induces a massive recall response, which reduces virus titers 2-3 days earlier than in nave controls. Influenza-specific mCTL produce interferon-gamma within 6 hr, but still take 4-5 days to localize to the infected respiratory tract. The delay reflects that the recall response develops first in the MLN, which contains relatively few mCTL. The response to a subdominant epitope is less obvious after secondary challenge.

Animals↗

PubMed1998

Individual variations in the murine T cell response to a specific peptide reflect variability in naive repertoires.

Previous studies have analyzed the diversity of T cell responses upon immunization. Little is known, however, about the individual variability of naive repertoires and its influence on immune responses. In the present study, T cells specific for a Kd-restricted epitope derived from HLA-A2 were purified from individual immunized mice using tetramers of MHC-peptide. Their TCRbeta chains were sequenced revealing strong biases but large variations in BJ usage and clonal composition. Most importantly, sequence analysis from nonimmunized mice demonstrated the preexistence of a small set of splenic precursors, distinct in each mouse and comprising less than 200 cells. Therefore, differences in precursor pools appear to be the major source of individual variability in antigen-selected repertoires.

Amino Acid Sequence↗

PubMed1998

Initiation of signal transduction through the T cell receptor requires the multivalent engagement of peptide/MHC ligands [corrected].

While much is known about intracellular signaling events in T cells when T cell receptors (TCRs) are engaged, the mechanism by which signaling is initiated is unclear. We have constructed defined oligomers of soluble antigen-major histocompatibility complex (MHC) molecules, the natural ligands for the TCR. Using these to stimulate specific T cells in vitro, we find that agonist peptide/MHC ligands are nonstimulatory as monomers and minimally stimulatory as dimers. Similarly, a partial-agonist ligand is very weakly active as a tetramer. In contrast, trimeric or tetrameric agonist ligands that engage multiple TCRs for a sustained duration are potent stimuli. Ligand-driven formation of TCR clusters seems required for effective activation and helps to explain the specificity and sensitivity of T cells.

Amino Acid Sequence↗

PubMed1997

The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the gammadelta T cell, G8.

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class 1b) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region-encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with beta2-microglobulin (beta2m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the gammadelta T cell clone, G8. Circular dichroism analysis indicates that T10/beta2m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.

Clone Cells↗

PubMed1997

Stability of empty and peptide-loaded class II major histocompatibility complex molecules at neutral and endosomal pH: comparison to class I proteins.

The structure and thermal stability of empty and peptide-filled forms of the murine class II major histocompatibility complex (MHC) molecule I-E(k) were studied at neutral and mildly acidic pH. The two forms have distinct circular dichroic spectra, suggesting that a conformational change may accompany peptide binding. Thermal stability profiles indicate that binding of peptide significantly increases the thermal stability of the empty heterodimers at both neutral and mildly acidic pH. Free energies calculated from these data provide a direct measure of this stabilization and show that the empty form of I-E(k) is significantly more stable than that of class I MHC proteins. Furthermore, for the two MHC class II proteins that were analyzed (I-E(k) and I-A(d)), thermal stability was not significantly altered by acidification. In contrast, of four class I MHC molecules studied, three have shown a significant loss in complex stability at low pH. The marked stability exhibited by their empty form, as well as their resistance to low pH, as observed in this study, correlate well with the ability of class II MHC molecules to traverse and bind peptides in acidic endosomal vesicles.

Amino Acid Sequence↗

PubMed1997

T cell receptor biochemistry, repertoire selection and general features of TCR and Ig structure.

T cell recognition is a central event in the development of most immune responses, whether appropriate or inappropriate (i.e. autoimmune). We are interested in reducing T cell recognition to its most elemental components and relating this to biological outcome. In a model system involving a cytochrome c-specific I-Ek restricted T cell receptor (TCR) derived from the 2B4 hybridoma, we have studied the interaction of soluble TCR and soluble peptide-MHC complexes using surface plasmon resonance. We find a striking continuum in which biological activity correlates best with the dissociation rate of the TCR from the peptide-MHC complex. In particular, we have found that weak agonists have significantly faster off-rates than strong agonists and that antagonists have even faster off-rates. This suggests that the stability of TCR binding to a given ligand is critically important with respect to whether the T cell is stimulated, inhibited or remains indifferent. It also suggests that the phenomenon of peptide antagonists might be explained purely by kinetic models and that conformation, either inter- or intramolecular, may not be a factor. We have also studied TCR repertoire selection during the establishment of a cytochrome c response, initially using an anti-TCR antibody strategy, but more recently using peptide-MHC tetramers as antigen-specific staining reagents. These tetramers work well with either class I or class II MHC-specific TCRs and have many possible applications. Lastly, we have also tried to correlate the structural and genetic features of TCRs with their function. Recent data on TCR structure as well as previous findings with antibodies suggest that both molecules are highly dependent on CDR3 length and sequence variation to form specific contacts with antigens. This suggests a general "logic' behind TCR and Ig genetics as it relates to structure and function that helps to explain certain anomalous findings and makes a number of clear predictions.

Animals↗

PubMed1996

Phenotypic analysis of antigen-specific T lymphocytes.

Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general, tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific for infectious agents, tumors, and autoantigens.

Amino Acid Sequence↗

PubMed1996

Calcium, magnesium, and phosphorus content of hair from two populations of rhesus monkeys.

The use of hair for the assessment of human mineral nutritional status is controversial, yet has potential because of the ease of collection and storage, and the high concentration of minerals in hair. This study generated baseline values for hair macromineral content in the most commonly used primate model for human nutrition and disease, the rhesus monkey. Hair digests from monkeys in Maryland (n = 98) and Puerto Rico (n = 208) were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). Significant differences existed between the two populations for hair calcium (Ca), magnesium (Mg), and phosphorus (P) (P < 0.01, P < 0.001, P < 0.02). Male hair had higher concentrations of Mg in specimens from the Puerto Rican population (P < 0.001), whereas hair phosphorus was higher in female hair (P < 0.02). There were no significant differences attributable to pregnancy status. Differences in hair content attributable to categorical age were found for Ca and Mg (P < 0.001, P < 0.01). Significant location differences paralleled dietary differences. These values therefore can be considered reference ranges for hair Ca, Mg, and P for free-ranging and caged rhesus monkeys.

Age Factors↗

PubMed1996

Copper, iron, manganese, and zinc content of hair from two populations of rhesus monkeys.

The concentrations of the elements copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) in hair of caged (in Maryland) and free-ranging (in Puerto Rico) rhesus monkey populations were determined. Significant chronological age-related decreases were evident for the trace elements Mn and Zn in both populations (P < or = 0.01) and for Cu and Fe in free-ranging monkeys (P < or = 0.005). The only overall gender difference was higher hair Mn concentration in free-ranging males than in females (P < 0.03). There were no significant differences in hair Cu, Fe, or Mn related to pregnancy status. Hair Zn was lower in samples from pregnant than from nonpregnant monkeys from the caged monkeys (P < or = 0.05), but did not differ in the free-ranging monkeys. Comparison of the two populations revealed that hair Zn levels were significantly higher in caged vs free-ranging animals (P < 0.001). We attribute this higher hair Zn in the caged monkeys to contact and ingestion of Zn from their galvanized enclosures. Hair iron levels were significantly higher (P < 0.001) in free-ranging monkeys, possibly as a result of Fe-rich soil ingestion from their environment. These data support continued investigation of the use of hair as an indicator of the exposure of large groups or populations to potentially high levels of specific minerals, and the use of nonhuman primates as models for the study of trace element deficiency and/or toxicity in humans.

Age Factors↗

PubMed1996

Tracking antigen-specific helper T cell responses.

T-cell receptor transgenic animals provide an excellent source of T cells for the analysis of antigen-specific helper T-cell development. Alternatively, studies in normal animals continue to focus on specific immune responses dominated by T cells using restricted sets of antigen receptors. These complementary strategies provide direct access to the dynamics of helper T-cell differentiation in vivo.

Animals↗

PubMed1996

Endothelium-dependent vasodilation in well-developed coronary collateral vessels.

We examined endothelium-dependent vasodilator responses in well-developed coronary collateral vessels. Collateral vessels and size-matched normal epicardial coronary arteries were isolated from canine hearts 6 months after a hollow plug had been introduced into the left anterior descending coronary artery (LAD) to stimulate collateral vessel growth. Vessel segments were isolated and studied in organ chambers. After preconstriction with prostaglandin F2 alpha, (PGF2 alpha) responses to the endothelium-dependent vasodilators, acetylcholine (ACh), substance P, and bradykinin (BK), as well as the endothelium-independent vasodilator nitroglycerin (NTG), were measured. The maximum relaxation to ACh was greater in collateral vessels (129 +/- 3.6% of the preconstricted tension) than in normal vessels (110 +/- 6.2%, p < 0.001), although the EC50 value for ACh was not significantly different between collateral and normal vessels. Collateral vessels demonstrated sensitivity to BK similar to that of normal vessels, but the maximal vasodilation achieved was greater in collaterals (138 +/- 4.6% of preconstricted tension) than in normal vessels (108 +/- 5.0%, p < 0.05). Both substance P and NTG produced dose-related relaxation that was similar in collateral vessels and normal arteries. These findings demonstrate that endothelium-dependent relaxation is intact in well-developed coronary collateral vessels.

Acetylcholine↗

PubMed1996

Enumeration and characterization of memory cells in the TH compartment.

There is a great deal of interest in understanding how helper T cells differentiate in vivo and exert their regulatory role on a developing, immune response. Essential to development of protective immunity is the development of memory T cells. To study memory T cells in vivo we first need the means to identify and characterize these cells as they develop in their complex microenvironments. We have developed a method which allows us to directly purify both primary and memory helper T cells from the draining lymph nodes of mice as they respond to pigeon cytochrome c in vivo. Junctional sequences from these populations and from individual T cells show a strong selection for CDR3 length and residues characteristic of antigen binding. Overall these studies support a model of progressive clonal maturation with the memory T cell repertoire being more homogeneous than that of the primary response. There is some suggestion that affinity maturation may take place after repeated immunization, but on a more modest scale than that seen for antibodies. Finally we present the use of two new technologies that promise to greatly expand the analysis of immune responses in vivo. The use of flow cytometry with simultaneous detection of five and six fluorescence parameters helps to reliably resolve rare subsets of antigen-specific cells in order to understand the progression of their differentiation in vivo. Lastly, we have developed peptide/MHC tetramers as a new class of staining reagent that has wide applicability in the study of T cell responses in vivo.

Amino Acid Sequence↗

PubMed1996

Vasomotor responses of newly developed coronary collateral vessels.

Well-developed coronary collateral vessels contain an abundant muscular media and can undergo active vasomotion. However, early after coronary occlusion, coronary collateral vessels are thin walled with little smooth muscle, suggesting that vasomotor capability might be limited. Consequently, this study determined whether newly developed coronary collateral vessels have active vasomotor activity and whether endothelial function in these newly developed vessels is impaired. Retrograde blood flow was measured as an index of coronary collateral blood flow approximately 2 wk after embolic occlusion of the anterior descending coronary artery of dogs. Agonists were administered into the left main coronary artery to reach collaterals originating from the left coronary system. Baseline retrograde blood flow was 25.1 +/- 2.7 ml/min and increased to 36.7 +/- 3.7 ml/min after nitroglycerin (6 micrograms.kg-1.min-1, P < 0.05). Cyclooxygenase blockade with indomethacin (5 mg/kg i.v.) decreased retrograde collateral blood flow to 16.8 +/- 2.3 ml/min (P < 0.05). Subsequent administration of acetylcholine increased retrograde flow to 29.4 +/- 3.7 ml/min (P < 0.05), indicating intact endothelium-mediated vasodilation. Inhibition of nitric oxide synthase with NG-nitro-L-arginine further decreased coronary collateral retrograde flow to 12.0 +/- 2.8 ml/min (P < 0.05) and markedly blunted the response to acetylcholine. These findings demonstrate substantial vasomotor capability even early during coronary collateral development and indicate that both nitric oxide and cyclooxygenase-dependent endothelial mechanisms are intact.

Animals↗