Production and characterization of T cell hybridomas.
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Animals↗
Biomedical subjects
P Marrack
Publications and source records attributed to P Marrack.
Activation changes the spectrum but not the diversity of genes expressed by T cells.
During activation T cells are thought to change their patterns of gene expression dramatically. To find out whether this is true for T cells activated in animals, the patterns of genes expressed in resting T cells and T cells 8 and 48 hr after activation were examined by using Affymetrix gene arrays. Gene arrays gave accurate comparisons of gene expression in the different cell types because the expression of genes known to vary during activation changed as expected. Of the approximately 6,300 genes assessed by the arrays, about one-third were expressed to appreciable extents in any of the T cells tested. Thus, resting T cells express a surprisingly large diversity of genes. The patterns of gene expression changed considerably within 8 hr of T cell activation but returned to a disposition more like that of resting T cells within 48 hr of exposure to antigen. Not unexpectedly, the activated T cells expressed genes associated with cell division at higher levels than resting T cells. The resting T cells expressed a number of cytokine receptor genes and some genes thought to suppress cell division, suggesting that the state of resting T cells is not a passive failure to respond to extant external stimuli.
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An inverse relationship between T cell receptor affinity and antigen dose during CD4(+) T cell responses in vivo and in vitro.
Multimeric peptide/class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptide-immunized C57BL/10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation either in vivo or in vitro.
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CD4+ T cell division in irradiated mice requires peptides distinct from those responsible for thymic selection.
We investigated the mechanism by which alpha/beta T cells expand upon transfer to T cell-deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester-labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4+ T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4+ T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4+ T cells in irradiated mice. These data establish that class II-bound peptides control the expansion of CD4+ T cells transferred to T cell-deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice.
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Proposed changes for NIH's Center for Scientific Review. Panel on Scientific Boundaries for Review. Center for Scientific Review Advisory Committee, National Institutes of Health.
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National Institutes of Health (U.S.)↗
Bystander virus infection prolongs activated T cell survival.
In animals, T cells often die rapidly after activation, unless activation occurs in the presence of inflammatory factors. To understand how such activated cells survive to participate in immune responses, we studied the effects of viral infection on T cells responding to an unrelated superantigen. Normal T cells activated by superantigen in uninfected mice died as a result of their activation, whereas T cells that were activated during vaccinia infection survived longer in vivo and in culture. This bystander effect of viral infection on activated T cells was independent of effects on the magnitude of the initial T cell response, on induction of Bcl-2 and Bcl-x, on T cell proliferation, and on Fas killing. The failure of such effects to predict the fate of activated T cells in vivo indicates that virus infections shape T cell responses via mechanisms that differ from those described previously. These mechanisms may contribute to the ability of viral infections to induce autoimmunity.
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Soluble class I MHC with beta2-microglobulin covalently linked peptides: specific binding to a T cell hybridoma.
Soluble forms of the mouse MHC class I molecule, Dd, were produced in which the peptide binding groove was uniformly occupied by peptides attached via a covalent flexible peptide linker to the N terminus of the associated beta2-microglobulin. The MHC heavy chain and beta2-microglobulin were firmly associated, and the molecules displayed an Ab epitope requiring proper occupancy of the peptide binding groove. Soluble Dd containing a covalent version of a well-characterized Dd-binding peptide from HIV stimulated a T cell hybridoma specific for this combination. Furthermore, a tetravalent version of this molecule bound specifically with apparent high avidity to this hybridoma.
Amino Acid Sequence↗
Type I interferons keep activated T cells alive.
Antigen injection into animals causes antigen-specific T cells to become activated and, rapidly thereafter, die. This antigen-induced death is inhibited by inflammation. To find out how inflammation has this effect, various cytokines were tested for their ability to interfere with the rapid death of activated T cells. T cells were activated in vivo, isolated, and cultured with the test reagents. Two groups of cytokines were active, members of the interleukin 2 family and the interferons (IFNs) alpha and beta. This activity of IFN-alpha/beta has not been described previously. It was due to direct effects of the IFNs on the T cells and was not mediated by induction of a second cytokine such as interleukin 15. IFN-gamma did not slow the death of activated T cells, and therefore the activity of IFN-alpha/beta was not mediated only by activation of Stat 1, a protein that is affected by both classes of IFN. IFN-alpha/beta did not raise the levels of Bcl-2 or Bcl-XL in T cells. Therefore, their activity was distinct from that of members of the interleukin 2 family or CD28 engagement. Since IFN-alpha/beta are very efficiently generated in response to viral and bacterial infections, these molecules may be among the signals that the immune system uses to prevent activated T cell death during infections.
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Live cell fluorescence imaging of T cell MEKK2: redistribution and activation in response to antigen stimulation of the T cell receptor.
T cell activation requires engagement of the T cell receptor (TCR) at the interface of conjugates formed with antigen-presenting cells. TCR engagement is accompanied by a redistribution of specific signaling molecules to the cytoplasmic region of the TCR complex. In this study, immunocytochemistry and live cell fluorescence imaging demonstrate that T cell MEK kinase 2 (MEKK2) is translocated to the T cell/antigen-presenting cell interface in response to antigen activation. MEKK2 translocation occurs more rapidly as the antigen concentration is increased. Biochemical activation of MEKK2 follows TCR stimulation, and expression of a dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation. Live cell fluorescence imaging thus enables characterization of signal transducers that are dynamically translocated following TCR engagement.
Androstadienes↗
Invariant chain can bind MHC class II at a site other than the peptide binding groove.
Invariant chain binds to class II molecules and guides them to the cell surface via the endosomes. Class II-associated invariant chain peptide (CLIP), a conserved sequence in an unstructured region of invariant chain, binds in the peptide binding groove of class II and is thought to be the major contributor to the interaction between invariant chain and class II molecules. However, other interaction sites between the two proteins may exist. The published data on this subject are conflicting. We have studied the ability of invariant chain to interact with a class II molecule in which the peptide binding groove of the protein is already occupied by a covalently attached peptide. Precipitation of these class II/peptide complexes with an Ab specific for this particular combination also precipitates invariant chain. This binding between class II/peptide and invariant chain is weak, and coprecipitation is only apparent in mild detergents. Thus, when the class II peptide binding groove is occluded by peptide and is not free to interact with CLIP, invariant chain can still bind the class II molecule at other lower affinity sites.
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T cell positive selection by a high density, low affinity ligand.
Interaction of the alpha beta T cell receptor (TCR) with major histocompatibility (MHC) molecules occupied with any of a large collection of peptides derived from self proteins is a critical step in driving T cell "positive" selection in the thymus. Interaction with this same pool of self-peptide/MHC ligands deletes T cells with potential self-reactivity. To examine how T cells survive both of these processes to form a self-tolerant mature repertoire, mice were constructed whose entire class II MHC IEk specific repertoire was positively selected on a single peptide covalently attached to the IEk molecule. In these mice T cells were identified that could respond to a variant of the positively selecting peptide bound to IEk. The affinities of the TCRs from these T cells for the positively selecting ligand were extremely low and at least 10-fold less than those for the activating ligand. These results support the theory that positive selection is driven by TCR affinities lower than those involved in T cell deletion or activation and that, if present at high concentration, even very low affinity ligands can positively select.
Amino Acid Sequence↗
Cytokine-induced survival of activated T cells in vitro and in vivo.
Many antigen-specific T cells die after exposure to antigen in animals. These cells also die if they are isolated from animals shortly after activation and cultured. Various cytokines were tested for their ability to interfere with this in vitro death. Surprisingly, tumor necrosis factor alpha and other inflammatory cytokines did not prevent the in vitro death of activated T cells, even though these cytokines do prevent activated T cell death in animals. Therefore, the inflammatory cytokines probably act on T cells in vivo via an intermediary factor. Four cytokines, interleukin (IL)-2, IL-4, IL-7, and IL-15, did prevent activated T cell death in vitro, with IL-4 and IL-15 more effective than IL-2 or IL-7. These cytokines share a component of their receptors, the common gamma chain, gammac. Therefore, their collective ability to protect activated T cells from death may be mediated by signals involving gammac. To assess their activity in vivo, two of the cytokines, IL-2 and IL-4, were expressed in animals at local sites of superantigen responses. Both cytokines increased the numbers of T cells found at the local sites 14 days later. Interleukin 4 was more effective than IL-2, even though IL-2 stimulates T cell proliferation better than IL-4. This result suggested that IL-4 and related cytokines can promote T cell survival in vivo as well as in vitro. The ability of these cytokines to prevent the death of activated T cells may be important at certain stages of immune responses in animals.
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Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes.
Multimeric soluble MHC class II molecules stably occupied with covalently attached peptides bind with appropriate specificity to T cell hybridomas and T cells from T cell receptor transgenic mice. There is a direct correlation between soluble T cell receptor affinity for monomeric MHC/peptide and level of binding of multimeric MHC/peptide to T cells. While binding of the multimeric MHC/peptide complex is proportional to T cell receptor affinity and expression level, there is little influence of T cell CD4.
Amino Acid Sequence↗
Crystal structure of mouse H2-M.
H2-M (HLA-DM in humans) resides in an acidic endosomal compartment, where it facilitates the loading of antigenic peptides into the peptide-binding groove of class II MHC. The crystal structure of a soluble form of H2-M has been solved to 3.1 A resolution, revealing a heterodimer with structural similarities to the MHC family of proteins. In contrast to its antigen-presenting cousins, the membrane distal alpha helices of H2-M pack closely together, occluding most of the binding groove except for a single large pocket near the center. The structure of H2-M has several unique features that may play a role in its function as a molecular chaperone and peptide exchange factor.
Amino Acid Sequence↗
T-cell survival.
Like other cells, T cells are dependent on signals from their environment for their survival. Resting T cells are supported in vitro by cytokines such as interleukin (IL)-4, IL-6 and IL-7. The latter two cytokines are made constitutively in animals and hence might affect the lifetimes of their resting T cells. Resting T cells are also kept alive by interaction with an as yet unidentified molecule on the surface of other cells. Activated T cells are also supported in vitro by members of two families of these proteins, the IL-2 family and the interferon-alpha beta family. Members of the latter family may have effects on activated cells in vivo. Thus although both resting and activated T cells require signals to keep themselves alive, the signals are different for the two types of cells. This perhaps allows the immune response to control the numbers of activated cells during infections without compromising its pool of precursor, resting T cells.
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Selection of antigen-specific T cells by a single IEk peptide combination.
In normal mice, major histocompatibility complex (MHC) proteins are bound to many different peptides, derived from the proteins of their host. In the thymus, the diversity of this collection of MHC + peptide ligands allows thymocytes bearing many different T cell receptors (TCRs) to mature by low avidity reactions between the MHC + peptide ligands and the thymocyte TCRs. To investigate this problem, the selection of T cells specific for a well-studied combination of MHC + peptide, IEk + moth cytochrome c 88-103 (MCC), was investigated. Mice were created that expressed IEk bound to a single peptide, either a variant of MCC in which a critical TCR contact residue, 99K, was changed to A, or a variant of a mouse hemoglobin 64-76 (Hb) peptide, 72A. IEk bound to the MCC variant caused the clonal deletion of some T cells specific for the IEk + MCC ligand; nevertheless, it also positively selected many T cells that could react with this ligand. Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not. IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC. These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.
Amino Acid Sequence↗
Interleukin 4 (IL-4) or IL-7 prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4.
Although much is known about the activation, proliferation, and function of CD4(+) T cells, little is known about how they survive as resting T cells in animals. Resting T cells have a half-life in animals of more than a week; however, when they are removed from animals and placed in tissue culture their half-life falls to approximately 24 h. In this paper, we show that the survival of resting T cells in vitro is promoted by two cytokines, interleukins 4 and 7 (IL-4, IL-7). They may do this in part by maintaining levels of survival-promoting proteins such as Bcl-2 in the cells, because the levels of Bcl-2 and Bcl-Xl in resting T cells fall rapidly after the cells are isolated from animals, and are maintained by culture in IL-4. Because the IL-4 receptor is known to signal through the JAK1 and JAK3/Stat6 pathway, we tested whether Stat6 was required for IL-4- dependent T cell survival. Surprisingly, we found that IL-4 rescued T cells from apoptosis in what appeared to be a Stat6-independent manner. These results demonstrate that the survival of resting T cells is an active process that can be affected by signals delivered by cytokines and also suggest that the IL-4 receptor on resting T cells may use a novel signaling pathway to facilitate T cell viability.
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IL-6 rescues resting mouse T cells from apoptosis.
It has previously been demonstrated that mature mouse T cells live for many weeks in vivo. In contrast, explanted lymph node or splenic T cells undergo spontaneous death within days, suggesting that survival factors supplied in vivo are not present in normal tissue culture medium. We discovered that IL-6 can rescue resting T cells from apoptosis in vitro. We show that recombinant mouse IL-6 as well as IL-6 in endothelial cell supernatants are sufficient to rescue T cells from death in the absence of additional cytokines. We show that CD4+ T cells express Bcl-2 immediately following isolation from the mouse, but after 24 h in culture Bcl-2 is undetectable. If during this time period the T cells are incubated with rIL-6, Bcl-2 expression is not down-regulated. It is, therefore, possible that IL-6 rescue from death is mediated by maintenance or induction of Bcl-2 expression. Addition of rIL-6 does not by itself induce blastogenesis or proliferation, and therefore, this cytokine appears to be a true survival factor rather than a mitogenic factor for resting T cells. Together, these results support a potential role for IL-6 as one of the factors important for prolonging resting T cell survival in vivo.
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