A novel POU domain protein which binds to the T-cell receptor beta enhancer.

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.

Jun kinase phosphorylates and regulates the DNA binding activity of an octamer binding protein, T-cell factor beta1.

POU domain proteins have been implicated as key regulators during development and lymphocyte activation. The POU domain protein T-cell factor beta1 (TCFbeta1), which binds octamer and octamer-related sequences, is a potent transactivator. In this study, we showed that TCFbeta1 is phosphorylated following activation via the T-cell receptor or by stress-induced signals. Phosphorylation of TCFbeta1 occurred predominantly at serine and threonine residues. Signals which upregulate Jun kinase (JNK)/stress-activated protein kinase activity also lead to association of JNK with TCFbeta1. JNK associates with the activation domain of TCFbeta1 and phosphorylates its DNA binding domain. The phosphorylation of recombinant TCFbeta1 by recombinant JNK enhances the ability of TCFbeta1 to bind to a consensus octamer motif. Consistent with this conclusion, TCFbeta1 upregulates reporter gene transcription in an activation- and JNK-dependent manner. In addition, inhibition of JNK activity by catalytically inactive MEKK (in which methionine was substituted for the lysine at position 432) also inhibits the ability of TCFbeta1 to drive inducible transcription from the interleukin-2 promoter. These results suggest that stress-induced signals and T-cell activation induce JNK, which then acts on multiple cis sequences by modulating distinct transactivators like c-Jun and TCFbeta1. This demonstrates a coupling between the JNK activation pathway and POU domain proteins and implicates TCFbeta1 as a physiological target in the JNK signal transduction pathway leading to coordinated biological responses.

Activation-induced T-cell death is cell cycle dependent and regulated by cyclin B.

Developing thymocytes and some T-cell hybridomas undergo activation-dependent programmed cell death. Although recent studies have identified some critical regulators in programmed cell death, the role of cell cycle regulation in activation-induced cell death in T cells has not been addressed. We demonstrate that synchronized T-cell hybridomas, irrespective of the point in the cell cycle at which they are activated, stop cycling shortly after they reach G2/M. These cells exhibit the diagnostic characteristics of apoptotic cell death. Although p34cdc2 levels are not perturbed after activation of synchronously cycling T cells, cyclin B- and p34cdc2-associated histone H1 kinase activity is persistently elevated. This activation-dependent induction of H1 kinase activity in T cells is associated with a decrease in the phosphotyrosine content of p34cdc2. We also demonstrate that transient inappropriate coexpression of cyclin B with p34cdc2 induces DNA fragmentation in a heterologous cell type. Finally, in T cells, cyclin B-specific antisense oligonucleotides suppress activation-induced cell death but not cell death induced by exposure to dexamethasone. We therefore conclude that a persistent elevation of the level of cyclin B kinase is required for activation-induced programmed T-cell death.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21(WAF1/Cip1) blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity.

Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21Cip1/Waf1/Sdi1 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase delta. The kinase inhibitory domain maps to the N-terminus (1-82) and contains the cdk2 binding site (28-82). We have generated a panel of deletion mutants of p21. A functional characterization of p21 mutants in the N-terminal domain reveals that cyclins bind to this domain independently of cdk2. Correlating with these results we find that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.

Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.

The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21.

Characterization of agretopes and epitopes involved in the presentation of beef insulin to T cells.

Beef insulin-specific I-Ad-restricted T cell hybridomas were derived from the fusion of antigen-primed (BALB/c X B6)F1 T cells with BW5147 thymoma. Specificity analysis revealed that the A-chain loop region is involved in antigen recognition. Hybridoma A20.2.15 is specific for beef insulin and cross-reacted with sheep insulin, but not with pork insulin. Using synthetic peptides we showed that the A-chain loop containing peptide A1-A14 jointed to the B7-B15 peptide by a disulfide bond can activate this hybridoma. Fragments generated by enzyme digest further suggest that the peptide recognized on beef insulin appears to involve A-chain loop residues A5-A12 and B-chain residues B7-B13 that are linked by the A7-B7 disulfide bridge. We found that beef insulin needs to be processed prior to T cell activation. Glutaraldehyde fixation and chloroquine treatment of presenting cells abolished their capacity to present insulin. Beef insulin denatured by pH changes cannot activate, thus suggesting that simple denaturation is not sufficient for presentation by antigen presenting cells. Finally, the agretope on beef insulin is comprised of two functional regions B7-B13 on the B chain and the A-chain loop in the A-chain, while residues A8 and A10 are probably involved in interaction with the T cell receptor.

Cloning of a T-cell receptor beta-chain enhancer binding protein.

Several eukaryotic DNA binding proteins have been isolated by screening lambda expression libraries with DNA probes containing their binding site. This strategy has been employed to isolate clones of the factor that interacts with the T-cell receptor beta-chain enhancer motif. A cDNA clone encoding a protein similar to YB-1 has been isolated with this. It seems probable that this protein YB, might interact with other proteins and regulate the transcription of the T-cell receptor beta-chain gene.

Immunological competence and host-specific tolerance of antibody-facilitated bone marrow chimeras.

We have generated murine antibody-facilitated (AF) bone marrow chimeras in the genetic combination P1----(P1 X P2)F1 by the simultaneous injection of P1 bone marrow cells and anti-P2 MHC monoclonal antibody into normal (unirradiated) adult (P1 X P2)F1 recipients. These mice have normal life spans and appear to be healthy, with no overt signs of graft-versus-host disease. We have undertaken an extensive survey of the ability of stable, long-term AF chimeras to generate immune responses in vitro and in vivo. Both T and B lymphocyte functions have been analyzed in proliferative and effector cell assays. The AF chimeras respond normally to mitogenic as well as antigenic stimuli, and exhibit normal capacities for cellular collaboration in the generation of immune responses. However, splenic lymphocytes from AF chimeras are substantially and specifically hyporesponsive or nonresponsive to host, P2-encoded, alloantigens in in vitro assays of cell-mediated immunity. This host-specific tolerance is exhibited by the cytotoxic T lymphocyte lineage; T helper cells necessary for the generation of a cytotoxic response may also have decreased reactivity to host determinants. We conclude that our protocol for the production of AF chimeras does not compromise the immune system of chimeric animals but does allow the maintenance of host-specific tolerance, after stable equilibrium has been attained.

Pancreatic islet transplantation: utility of ductular obstruction and exocrine atrophy model?

Introduction of 'silent' exocrine atrophy (and endocrine 'enrichment') in pancreatic grafts following ductular blockade may have a role in human diabetes by circumventing currently elusive islet isolation/purification protocols. To explore this potential, pancreatic isografts were performed in 12 pairs of inbred Wistar NIN rats. Donor pancreatectomy was performed after distal clamping and canulation of common bile duct and injection of 0.5 ml. polyacrylamide gel (blocked n = 7) or normal saline (un-blocked n = 5) respectively. One to 2 m.m. fragments of the resulting mildly distended pancreases were transplanted in to 2 sites (renal capsule and iliac fossa subcutaneously) of cach recipient. Post-operative biopsies of the transplanted grafts (unilateral nephrectomy and iliac fossa biopsies) revealed macroscopic and microscopic evidence of necrotizing pancreatitis in both the groups at both the sites (histiocytic and giant cell infiltration, fat necrosis and focal calcification with destruction of exocrine and endocrine cells) as early as 1 and 3 weeks. Possible detrimental factors include: volume and pressure of ductal injection, graft sites (confined spaces), post-operative wound infection and bio-compatibility of the material used for ductular blockade.

Cell cycle control of DNA replication.

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (cdk), which phosphorylate and activate proteins that execute events critical to cell cycle progression. In mammalian cells cdk2-cyclin A has a role in S phase. Many replication proteins are potential substrates for this cdk kinase, suggesting that initiation, elongation and checkpoint control of replication could all be regulated by cdk2. The association of PCNA, a replication protein, with cdk-cyclins during G-1 to S phase transition and with cdk-cyclin inhibitors, adds an interesting complexity to regulation of DNA replication.

Apoptosis and the cell cycle.

Apoptosis is a genetically controlled response by which eukaryotic cells undergo programmed cell death. This phenomenon plays a major role in developmental pathways (1), provides a homeostatic balance of cell populations, and is deregulated in many diseases including cancer. Control of cell number is determined by an intricate balance of cell death and cell proliferation. Accumulation of cells through suppression of death can contribute to cancer and to persistent viral infections, while excessive death can result in impaired development and in degenerative diseases. Identification of genes that control cell death, and understanding of the impact of apoptosis in both development and disease has advanced our knowledge of apoptosis in the past few years. There appears to be a linkage between apoptosis and cell cycle control mechanisms. Elucidating the mechanisms that link cell cycle control with apoptosis will be of key importance in understanding tumour progression and designing new models of effective tumour therapy.

The functions of the cdk-cyclin kinase inhibitor p21WAF1.

p21WAF1 plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21WAF1 (hereafter referred to as p21) are the cdk-cyclins which regulate the progression of eukaryotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase delta. p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit DNA synthesis. These distinct properties map to the N-terminal and the C-terminal regions of p21, respectively. Cell cycle arrest in G-1 (G-1 checkpoint) following DNA damage is mediated by p53 and is deficient in p21 null cells. p53 thus upregulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity. Retinoblastoma protein is regulated by cdk-cyclin kinases, and acts as a downstream target of p21 in DNA damage-induced G-1 arrest. Furthermore, accumulating evidence indicates that p21 may play a role in maintaining G-2 arrest after DNA damage. Transcriptional control of p21 by factors other than p53 is critical for growth arrest and for cell differentiation in many instances.

Proteolytic cleavage of retinoblastoma protein upon DNA damage and Fas-mediated apoptosis.

Proteolytic cleavage of key cellular proteins by caspases (ICE, CPP32, and Ich-1/Nedd2) may be crucial to the apoptotic process. The retinoblastoma tumor suppressor gene is a negative regulator of cell growth and the retinoblastoma protein (pRb) exhibits anti-apoptotic function. We show that pRb is cleaved during apoptosis induced by either UV irradiation or anti-Fas antibody. Our studies implicate CPP32-like activity in the proteolytic cleavage of pRb. The kinetics of proteolytic cleavage of pRb during apoptosis differ from that observed for other cellular proteins, suggesting that the specific cleavage of pRb during apoptosis may be an important event.

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas.

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.

Lepromin conversion in repeatedly lepromin negative BL/LL patients after immunization with autoclaved Mycobacterium w.

Thirty-two clinically, histopathologically confirmed cases of BL/LL leprosy were rendered bacteriologically negative by prolonged chemotherapy. All of them were negative to Mitsuda and Dharmendra lepromin at the start of study. They were immunized with a single intradermal injection of 5 X 10(7) autoclaved Mycobacterium w and were retested for lepromin reaction 4-6 weeks later. Twenty subjects gave at this time a positive reaction with both Dharmendra and Mitsuda lepromins. The histology of biopsies from converted cases showed mononuclear infiltration in all and granuloma formation in 12 of the 20 positive cases. The stability of the conversion of the patients' lepromin positivity was investigated 6-11 months after immunization with Mycobacterium w. Patients who were earlier converted to a positivity status remained positive in the skin test response to M. leprae. The leukocytes of these patients produced lymphokines on culture with lepromin, causing leukocyte migration inhibition. Patients who did not convert earlier continued to remain anergic to lepromin. These results suggest a conversion, stable for several months, to lepromin positivity caused by immunization with Mycobacterium w in about 60% of BL/LL leprosy patients.

Gene conversion may be responsible for the generation of the alloreactive repertoire.

Meiotic gene conversion in the major histocompatibility complex (MHC) appears to be involved in the generation of MHC polymorphism. Here the authors suggest that mitotic gene conversion of MHC may generate somatic variants which sensitize the immune system. This could lead to an MHC-skewed repertoire with a high background alloreactive potential crossreacting with foreign antigens in the context of self MHC. Allodeterminant-bearing autologous cells may be deleted by the immune cells, thereby maintaining the observed genotype/phenotype correlation.