Regulation of heat-induced apoptosis by Mcl-1 degradation and its inhibition by Hsp70.

Cellular stress eliminates irreversibly damaged cells by initiating the intrinsic death pathway. Cell stress is sensed by pro- and antiapoptotic members of the Bcl-2 protein family, which regulate the release of apoptogenic factors, such as cytochrome c, from mitochondria. Exposure of cells to hyperthermia results in the activation of the proapoptotic Bcl-2 family protein Bax, which plays an essential role in cytochrome c release. Heat directly affects Bax activity in vitro; however, antiapoptotic Bcl-2 family proteins, such as Bcl-xL, can suppress this activation, suggesting that a second heat-sensitive step must be breached before apoptosis ensues in cells exposed to hyperthermia. Here we show that heat shock causes the loss of Mcl-1 protein. Depletion of Noxa by short hairpin RNA protected cells from hyperthermia by preventing Mcl-1 degradation. Heat shock caused the dissociation of Noxa from Mcl-1, which allowed binding of the BH3-containing ubiquitin ligase Mule followed by Mcl-1 ubiquitination and degradation. Overexpression of Hsp70, which prevents heat-induced Bax activation, stabilized Mcl-1 protein levels in heat-shocked cells. This resulted from reduced Mule binding and ubiquitination as well as enhanced Mcl-1 expression compared with cells without Hsp70. Our results demonstrate that loss of Mcl-1 is a critical heat-sensitive step leading to Bax activation that is controlled by Hsp70.

Dendritic cells need T cell help to prime cytotoxic T cell responses to strong antigens.

Peptide-pulsed mouse dendritic cells (DC) primed peptide-specific CD8+ cytotoxic T cell responses very effectively if they expressed minor histocompatibility antigens, which could stimulate a CD4+ T helper cell response. These DC could also prime most syngeneic mice, although there was no deliberate immunization for help (the DC were prepared in syngeneic mouse serum, to avoid any response to fetal calf serum antigens). In contrast, DC were unable to prime MHC class II-deficient mice for cytotoxic responses to the classical helper-dependent antigens Qa1a and HY. More strikingly, Balb.B DC failed to prime B6 MHC class II-deficient mice for cytotoxic responses to Balb minor antigens, even though these two strains differ at more than 40 minor histocompatibility loci. When peptide-pulsed DC were prepared without enzymes (used to release DC from lymphoid tissues), they failed to prime the majority of normal syngeneic mice, even though they expressed high levels of B7 and ICAM-1 co-stimulatory molecules, suggesting that help was provided by responses to antigens in the enzyme cocktail. The enzyme treatment itself did not provide signals that could substitute for help, since DC prepared with enzymes could not prime MHC class II-deficient mice. The observation that highly immunogenic minor-incompatible DC failed to prime MHC class II-deficient mice suggests that in the absence of inflammatory signals, even strong antigens cannot stimulate CD8+ T cell responses without help.

Antigen processing in populations of mature murine dendritic cells is caused by subsets of incompletely matured cells.

Immature dendritic cells (DC), such as freshly isolated Langerhans cells (LC), are excellent at processing native protein Ag. During short term culture they shut off MHC class II synthesis and down-regulate their processing capacity. They retain, however, the MHC/peptide complexes, up-regulate adhesion and costimulatory molecules, and acquire the ability to sensitize T cells. Two reports describing substantial processing activity in populations of mature DC prompted us to undertake an extensive comparative study of the Ag-processing capacities of immature vs mature DC. We used a panel of 17 peptide-specific T cell hybridomas restricted by six different MHC class II molecules: I-Ab, I-A(d), I-E(d), hybrid I-A beta dE alpha, I-Ak, and I-Ek. Side by side comparisons revealed in all cases that freshly isolated LC were superior to cultured mature LC in their ability to process native proteins. With some hybridomas, however, we found a considerable degree of processing by populations of cultured LC at high doses of Ag or Ag-presenting cells. This activity, however, did not correlate with the MHC haplotype. Direct comparison over wide ranges of DC doses or Ag doses showed that it was always less than that of corresponding fresh immature LC. Immunoperoxidase staining of cytospins and flow cytometry with mAb In1 disclosed a small (20% maximum) subset of cultured LC expressing the MHC class II-associated invariant chain, indicating ongoing biosynthesis of this molecule and, thus, incomplete maturation of these LC. Therefore, the residual processing activity observed in populations of mature DC may be explained by small subpopulations of incompletely matured DC.

The T cell receptor repertoire influences V beta element usage in response to myoglobin.

T cell clones recognizing the sperm whale myoglobin (SpWMb) epitope 110-121 in association with H-2d major histocompatibility complex class II molecules display a very limited heterogeneity of T cell receptor (TCR) V beta usage in DBA/2 mice. All clones previously tested used the same V beta 8.2 gene segment and very restricted junctional regions. To investigate the significance of this observation in vivo, we immunized DBA/2 mice with the intact SpW Mb protein or peptide 110-121. Only the V beta 8+ T cells showed any significant response to the 110-121 epitope. The response to peptide 110-121 was then analyzed in mice which, either as a consequence of antibody depletion or through genetic deletion of TCR V beta genes, lacked V beta 8+ peripheral T cells. DBA/2 mice depleted of V beta 8+ T cells by antibody treatment responded poorly to the 110-121 peptide, and only at high antigen concentrations. In contrast, DBA/2V beta a mice (homozygous for a deletion of multiple V beta gene segments including the V beta 8 family) made a response at least as great as that made by DBA/2 mice, even though the DBA/2V beta a mice had a very restricted TCR V beta repertoire compared with DBA/2 mice. Mechanisms which might determine differences in the 110-121 specific response of DBA/2, DBA/2V beta a and F23.1-treated DBA/2 mice are discussed.

Cim: an MHC class II-linked allelism affecting the antigenicity of a classical class I molecule for T lymphocytes.

Two alleles at the major histocompatibility complex (MHC)-linked locus cim determine "gain and loss" changes in the rat RT1.Aa class I molecule which affect its structure both as an alloantigen and as a restriction element. Alleles at the cim locus also influence the post-translational modification of RT1.Aa. These effects may reflect the participation of the cim gene product in the processes of peptide loading or assembly of RT1.Aa. In this study we have used the discriminating RT1.Aa-specific monoclonal antibody JY3/84, as well as cytotoxic T cells raised in appropriate combinations, to determine the cim alleles of eight haplotypes in 15 independent inbred strains of rat. We have also employed the same techniques to analyse a panel of F1 hybrid animals derived from various MHC recombinant strains. These experiments map the cim locus to the class II region of RT1, probably between the DP-related genes (RT1.H) and the DQ-related RT1.B alpha.

The presumptive CDR3 regions of both T cell receptor alpha and beta chains determine T cell specificity for myoglobin peptides.

The T cell receptor alpha/beta (TCR-alpha/beta) is encoded by variable (V), diversity (D), joining (J), and constant (C) segments assembled by recombination during thymocyte maturation to produce a heterodimer that imparts antigenic specificity to the T cell. Unlike immunoglobulins (Igs), which bind free antigen, the ligands of TCR-alpha/beta are cell surface complexes of intracellularly degraded antigens (i.e., peptides) bound to and presented by polymorphic products of the major histocompatibility complex (MHC). Therefore, antigen recognition by T cells is defined as MHC restricted. A model has been formulated based upon the similarity between TCR-alpha/beta V region and Ig Fab amino acid sequences, and the crystal structure of the MHC class I and Ig molecules. This model predicts that the complementarity determining regions (CDR) 1 and 2, composed of TCR V alpha and V beta segments, primarily contact residues of the MHC alpha helices, whereas V/J alpha and V/D/J beta junctional regions (the CDR3 equivalent) contact the peptide in the MHC binding groove. Because polymorphism in MHC proteins is limited relative to the enormous diversity of antigenic peptides, the TCR may have evolved to position the highly diverse junctional residues (CDR3), where they have maximal contact with antigen bound in the MHC peptide groove. Here, we demonstrate a definitive association between CDR3 sequences in both TCR alpha and beta chains, and differences in recognition of antigen fine specificity using a panel of I-Ed-restricted, myoglobin-reactive T cell clones. Acquisition of these data relied in part upon a modification of the polymerase chain reaction that uses a degenerate, consensus primer to amplify TCR alpha chains without foreknowledge of the V alpha segments they utilize.

A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule.

The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post-translational modification of RT1.Aa.

Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells.

The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.

Correlation of T cell receptor V beta gene family with MHC restriction.

We have studied a panel of DBA/2 T cell clones specific for sperm whale myoglobin (SpW Mb) for TCR (T cell receptor) beta chain gene expression by FACS analysis using the monoclonal antibodies F23.1 and KJ16 specific for the V beta 8 family of the TCR beta chain genes. Within any given specificity group, all the clones tested came from different mice. 10 of 11 I-Ed-restricted SpW Mb-specific T cell clones were F23.1+; 8 of these were also KJ16+. Only one of the three I-Ad-restricted clones tested was F23.1+; this clone was KJ16 negative. This study has demonstrated that I-Ed-restricted T cell clones from DBA/2 mice express members of the TCR V beta 8 family irrespective of the epitopes of SpW Mb recognized. These data suggest an apparent correlation between TCR V beta expression and MHC restriction.

The structure of T-cell epitopes.

We have reviewed here studies using synthetic peptides to analyze some of the properties of T-cell epitopes. Several general conclusions can be drawn. First, T-cell epitopes can usually be defined by linear sequences of about seven amino acids. However, the observation that increasing peptide length often results in increased antigenic potency has suggested that antigenicity may crucially depend upon the ability of peptides to adopt appropriate secondary structures. Two models for the prediction of T-cell epitopes on the basis of primary sequence data alone were discussed. Biophysical studies on the association of peptides with Ia molecules have shown that antigenic peptides bind directly to Ia; the evidence suggests that a binary association between Ia and peptide occurs in the absence of specific T-cells. Finally, a hypothesis to explain the observation that B-cells and T-cells generally recognize distinct epitopes on multideterminant antigens has been examined.