Hepatitis C virus replication in mice with chimeric human livers.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Cytoplasmic processing is a prerequisite for presentation of an endogenous antigen by major histocompatibility complex class II proteins.

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of select epitopes derived from cytoplasmic antigens, with few insights into the processing reactions necessary for this alternate pathway. Efficient presentation of an immunodominant epitope derived from glutamate decarboxylase (GAD) was observed regardless of whether this antigen was delivered exogenously or via a cytoplasmic route into human histocompatibility leukocyte antigen class II-DR4(+) antigen-presenting cells. Presentation of exogenous as well as cytoplasmic GAD required the intersection of GAD peptides and newly synthesized class II proteins. By contrast, proteolytic processing of this antigen was highly dependent upon the route of antigen delivery. Exogenous GAD followed the classical pathway for antigen processing, with an absolute requirement for endosomal/lysosomal acidification as well as cysteine and aspartyl proteases resident within these organelles. Presentation of endogenous GAD was dependent upon the action of cytoplasmic proteases, including the proteasome and calpain. Thus, translocation of processed antigen from the cytoplasm into membrane organelles is necessary for class II-restricted presentation via this alternate pathway. Further trimming of these peptides after translocation was mediated by acidic proteases within endosomes/lysosomes, possibly after or before class II antigen binding. These studies suggest that processing of exogenous and cytoplasmic proteins occurs through divergent but overlapping pathways. Furthermore, two cytoplasmic proteases, the proteasome and calpain, appear to play important roles in MHC class II-restricted antigen presentation.

Expression of T cell receptor genes in an antigen-specific hybridoma and radiation-induced variants.

We have analyzed a series of mutants derived from a KLH-specific, I-E-restricted T hybridoma (FN1-18) which have lost antigen-reactivity while retaining both T cell receptor idiotypic determinants and the ability to respond to Con A. The variants have not gained any detectable alloreactivity, nor is there an obvious lesion in the mutants' beta chain DNA containing the utilized beta chain genes. This loss of antigen reactivity is due to a failure of stable production of the specific V beta-containing mRNA. Our results indicate that in FN1-18, the T cell receptor antigenic determinants are most likely carried by the alpha chain alone or by a complementation product of the V alpha FN1-18 with the V beta of BW5147. V beta FN1-18 represents a previously undescribed T cell receptor V region.

Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain.

In the polymerase chain reaction (PCR), two specific oligonucleotide primers are used to amplify the sequences between them. However, this technique is not suitable for amplifying genes that encode molecules where the 5' portion of the sequences of interest is not known, such as the T cell receptor (TCR) or immunoglobulins. Because of this limitation, a novel technique, anchored polymerase chain reaction (A-PCR), was devised that requires sequence specificity only on the 3' end of the target fragment. It was used to analyze TCR delta chain mRNA's from human peripheral blood gamma delta T cells. Most of these cells had a V delta gene segment not previously described (V delta 3), and the delta chain junctional sequences formed a discrete subpopulation compared with those previously reported.

Induction of interleukin 2 messenger RNA inhibited by cyclosporin A.

Cyclosporin A blocked production of the lymphokine interleukin 2 by activated T lymphocytes. In a human and a murine cell line this inhibition reflected an absence of interleukin 2 messenger RNA. Under conditions in which these cells are normally stimulated to secrete high levels of interleukin 2, they failed to do so in the presence of cyclosporin A. In both cell lines this failure was accompanied by an absence of interleukin 2 messenger accumulation.

Expression of T cell antigen receptor heterodimers in a lipid-linked form.

The interaction of the T cell receptor for antigen (TCR) with its antigen-major histocompatibility complex ligand is difficult to study because both are cell surface multimers. The TCR consists of two chains (alpha and beta) that are complexed to the five or more nonpolymorphic CD3 polypeptides. A soluble form of the TCR was engineered by replacing the carboxyl termini of alpha and beta with signal sequences from lipid-linked proteins, making them susceptible to enzymatic cleavage. In this manner, TCR heterodimers can be expressed independently of the CD3 polypeptides and in significant quantities (0.5 milligram per week). This technique seems generalizable to biochemical and structural studies of many other cell surface molecules as well.

Large scale isolation, growth, and function of porcine neonatal islet cells.

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice.

Naturally processed T cell epitopes from human glutamic acid decarboxylase identified using mice transgenic for the type 1 diabetes-associated human MHC class II allele, DRB1*0401.

The identification of class II binding peptide epitopes from autoimmune disease-related antigens is an essential step in the development of antigen-specific immune modulation therapy. In the case of type 1 diabetes, T cell and B cell reactivity to the autoantigen glutamic acid decarboxylase 65 (GAD65) is associated with disease development in humans and in nonobese diabetic (NOD) mice. In this study, we identify two DRB1*0401-restricted T cell epitopes from human GAD65, 274-286, and 115-127. Both peptides are immunogenic in transgenic mice expressing functional DRB1*0401 MHC class II molecules but not in nontransgenic littermates. Processing of GAD65 by antigen presenting cells (APC) resulted in the formation of DRB1*0401 complexes loaded with either the 274-286 or 115-127 epitopes, suggesting that these naturally derived epitopes may be displayed on APC recruited into pancreatic islets. The presentation of these two T cell epitopes in the islets of DRB1*0401 individuals who are at risk for type 1 diabetes may allow for antigen-specific recruitment of regulatory cells to the islets following peptide immunization.

Long-term prevention of diabetes and marked suppression of insulin autoantibodies and insulitis in mice lacking native insulin B9-23 sequence.

We analyzed double native insulin gene knockout NOD mice with a mutated (B16:alanine) proinsulin transgene at multiple ages for the development of insulin autoantibodies, insulitis, and diabetes. In contrast to mice with at least one copy of a native insulin gene that expressed insulin antibodies, only 2 out of 21 (10%) double native insulin gene knockout mice with a mutated insulin transgene developed insulin autoantibodies. Of 21 double insulin knockout mice sacrificed between 10 to 48 weeks of age, only 5 showed minimal insulitis versus 100% of wild-type NOD and more than 90% of insulin 1 knockout mice. Consistent with robust suppression of insulin autoantibodies and insulitis, no double insulin knockout mice developed diabetes. In that the B9-23 peptide with B16A is an altered peptide ligand inducing Th2 responses, we analyzed transfer of splenocytes into NOD.SCID mice. There was no evidence for regulatory T cells able to inhibit transfer of diabetes by diabetogenic NOD splenocytes. Insulin peptide B9-23 is likely a crucial target for initiation of islet autoimmunity and further mutation of the sequence will be tested to attempt to eliminate all anti-islet autoimmunity.

Advantages in the use of L-asparaginase-albumin polymer as an antitumor agent.

Polymeric conjugates of L-asparaginase and an excess of homologous albumin were compared with free L-asparaginase for antitumor activity using a mouse model 6C3HED lymphosarcoma and a human pancreatic tumor cell line, PANC-1. The asparaginase-albumin polymer is more resistant to proteolytic degradation compared to equivalent amounts of free enzyme and is more effective as an antitumor agent in prolonging survival of C3H/HeJ mice receiving 6C3HED lymphosarcoma. In terms of antitumor activity, the enzyme is approximately 20 times more effective, compared to free enzyme, when given in polymeric form with albumin. Similarly, the polymeric form of L-asparaginase is more effective in inhibiting cell growth of human pancreatic tumor cells grown in tissue culture. The increased effectiveness of the polymeric form of L-asparaginase is probably related to its resistance to biodegradation. The use of cell surface-specific monoclonal antibodies to target the polymer to tumor cells is also demonstrated.

Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression.

We prepared single-cell suspensions of Lewis rat ¿RT1(1/l)¿ testicular cells and cultured these in vitro for 48 h under conditions that promoted the formation of cellular aggregates. In the absence of systemic immunosuppression, the transplantation of a sufficient quantity of these aggregates (containing 11 x 10(6) cells, (75% Sertoli cells), together with 2,000 purified Lewis rat islets, reversed the diabetic state for >95 days in 100% (5/5) of the chemically diabetic Wistar-Furth ¿RT1(u/u)¿ recipients. Similar grafts consisting of islets alone or islets plus 50% fewer testicular cell aggregates survived for only 10 days. Functioning composite allografts harvested from normoglycemic animals at approximately 100 days showed healthy beta-cells in close association with Fas ligand-expressing Sertoli cells. Because no gene therapy protocol is required, the transplantation of composite grafts consisting of purified human allogeneic islets plus human allogeneic testicular cell aggregates can be applied in clinical islet transplantation as soon as it has been proven in a large animal model.

Expression of Gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis.

Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.

Retardation or acceleration of diabetes in NOD/Lt mice mediated by intrathymic administration of candidate beta-cell antigens.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

Beta-cell destruction in NOD mice correlates with Fas (CD95) expression on beta-cells and proinflammatory cytokine expression in islets.

A mechanism of autoimmune destruction of islet beta-cells in type 1 diabetes has been proposed to be the binding of Fas ligand (FasL) on T-cells to Fas receptors on beta-cells. We investigated this proposal by examining the expression of FasL and Fas on islet-infiltrating T-cells and beta-cells in relation to beta-cell destruction in a syngeneic islet transplant model in NOD mice. Diabetic NOD mice were transplanted with syngeneic islets and injected with complete Freund's adjuvant, which prevented diabetes recurrence (nondestructive insulitis), and with phosphate-buffered saline, which did not (beta-cell destructive insulitis). Two-color immunohistochemical assays revealed that FasL was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from both diabetic and normoglycemic mice, and the percentage of each type of cell that expressed FasL was greater in islet grafts from normoglycemic compared with diabetic mice. In contrast, Fas was expressed on CD4+ T-cells, CD8+ T-cells, and beta-cells in islet grafts from diabetic mice, but it was nearly or totally absent on these cells in islet grafts from normoglycemic mice. Similarly, polymerase chain reaction analysis of islet grafts revealed that Fas mRNA expression was significantly lower in islet grafts from normoglycemic compared with diabetic mice. Also, mRNA levels of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma were significantly lower in islet grafts from normoglycemic mice. Finally, Fas was induced on NOD islet cells by incubation with IL-1beta, IFN-gamma, and the combination of IL-1beta, TNF-alpha, and IFN-gamma. These findings support the concept that cytokine-induced Fas receptor expression on islet beta-cells is a mechanism for their destruction by FasL-expressing CD4+ and CD8+ T-cells and, possibly, by FasL-expressing beta-cells themselves.

Neonatal porcine islet cells induce human CD4+, but not CD8+, lymphocyte proliferation and resist cell-mediated cytolytic injury in vitro.

Xenotransplantation of porcine tissue to human recipients promises to alleviate the organ shortage. Human antibody-mediated and cell-mediated immune responses against porcine grafts, however, represent barriers to successful xenotransplantation. We compared neonatal porcine islet cells (NPICs) and neonatal porcine splenocytes for the ability to stimulate proliferation of human peripheral blood lymphocytes (PBLs), and for their susceptibility to human natural killer (NK) and cytotoxic T-lymphocyte (CTL)-mediated lysis. Human peripheral blood CD4+ lymphocytes showed strong proliferation in response to NPICs, likely because of occasional swine leukocyte antigen (SLA) class II+ cells in the NPIC preparations. In contrast, human peripheral blood CD8+ lymphocytes did not proliferate in response to NPICs, although they showed clear responses to both porcine splenocytes and endothelial cells. Both human CTL-raised-against-porcine splenocytes and endogenous NK cells lysed porcine splenocytes, but the same cells showed little or no lytic activity against NPICs. Lysis of porcine splenocyte targets was completely abrogated by pretreatment of the human NK or CTL populations with concana-mycin A, suggesting a perforin-dependent effector mechanism. Pretreatment of the NPIC targets with proinflammatory porcine cytokines to upregulate SLA class I expression failed to enhance human CTL-mediated lysis. However, lysis of NPICs by human CTLs could be elicited when a lectin was added to form stable effector:target cell conjugates. It appears that NPICs do not express sufficiently high levels of co-stimulatory and/or adhesion molecules to either activate human CD8+ T-cells or to be effective targets for activated human CTLs. These data suggest that NPICs may not be destroyed by NK- or CTL-mediated lytic mechanisms after transplantation into humans.

Major DQ8-restricted T-cell epitopes for human GAD65 mapped using human CD4, DQA1*0301, DQB1*0302 transgenic IA(null) NOD mice.

The 65KD isoform of GAD is considered to be a major target autoantigen in many humans with autoimmune prediabetes or diabetes. The major histocompatibility complex class II allele DQA1*0301, DQB1*0302, which encodes HLA-DQ8, confers susceptibility to type 1 diabetes and occurs in up to 80% of affected individuals. To map T-cell epitopes for GAD65 restricted to the diabetes-associated DQ8 heterodimer, we generated transgenic NOD mice expressing HLA-DQ8 and human CD4 while having the mouse class II gene (IA(beta)) deleted. These mice were immunized with full-length purified recombinant GAD65, and the fine specificity of T-cell responses was mapped by examining recall responses of bulk splenocytes to an overlapping set of 20-mer peptides encompassing the entire GAD65 protein. Four different peptides (P121-140, P201-220, P231-250, and P471-490) gave significant T-cell recall responses. P201-220 and P231-250 have been shown previously to bind DQ8, whereas the other two peptides had been classified as nonbinders. Interestingly, the peptide giving the greatest response (P201-220) encompasses residues 206-220 of GAD65, a region that has been shown to be a dominant T-cell epitope in wild-type IA(g7) NOD mice. Overlap in this T-cell epitope likely reflects structural similarities between DQ8 and IA(g7). The fine specificity of antibody responses in the GAD65-immunized mice was also examined by testing the antisera by enzyme-linked immunosorbent assay (ELISA) against the same overlapping set of peptides. The two dominant B-cell epitopes were P361-380 and P381-400; P121-140 and P471-490 appeared to correspond to both B- and T-cell epitopes. Although the NOD human CD4, DQ8, IA(null) transgenic mice generated in these studies do not develop autoimmune diabetes either spontaneously or after cyclophosphamide treatment, they can be used to map DQ8-restricted T-cell epitopes for a variety of human islet autoantigens. They can also be used to test T-cell-specific reagents, such as fluorescently labeled DQ8 tetramers containing GAD65 peptides or other beta-cell peptides, which we believe will be useful in analyzing human immune responses in diabetic and prediabetic patients.

Immunization with the larger isoform of mouse glutamic acid decarboxylase (GAD67) prevents autoimmune diabetes in NOD mice.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) has been implicated in autoimmune diabetes in NOD mice, but the role of the 67-kDa GAD isoform (GAD67) is less clear. We found that immunization of 4-week-old NOD mice with purified recombinant mouse GAD67 prevented or significantly delayed the onset of diabetes. To further explore this phenomenon, we characterized anti-GAD67 immune responses in naive and GAD-immunized NOD mice. Anti-GAD67 antibodies titers were relatively low in naive mice at all ages, but a single immunization with GAD67 at 4 weeks induced high titers of anti-GAD antibodies by 6 weeks of age. In both 4-week-old and diabetic NOD mice, there were significant endogenous T-cell proliferative responses against purified recombinant mouse GAD67. These T-cell proliferative responses were blocked by anti-I-ANOD and anti-CD4 antibodies. To characterize the anti-GAD T-cell responses in the NOD mice, we established T-cell lines and T-cell clones which recognized GAD67, and we used recombinant subfragments of GAD to localize the predominant T-cell epitopes in GAD67. T-cells from naive NOD mice proliferated in response to all GAD subfragments, whereas T-cells from diabetic mice responded primarily to the COOH-terminal 83 amino acids of GAD67. These results suggest that GAD67 is an autoantigen in IDDM and immunization of prediabetic NOD mice with GAD67 can prevent the onset of diabetes.

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Alternatively spliced, germline J alpha 11-2-C alpha mRNAs are the predominant T cell receptor alpha transcripts in mouse kidney.

We recently reported the expression of a truncated T cell receptor (TCR) alpha mRNA in kidney and brain of normal mice. In the kidney, the truncated TCR alpha transcript was expressed by bone marrow-dependent, non-T large interstitial cells located predominantly in the medulla. Here, we report the molecular characterization of the truncated TCR alpha transcript from kidney. Using a modified anchored-PCR (A-PCR) technique and directional cloning, 37 cDNA clones extending 5' of the C alpha region were generated. cDNA sequencing showed that 29 of the clones (78%) originated in the J alpha 11-2 region. Of these clones, 17 started upstream or in the J alpha 11-2 exon and contained the entire J alpha 11-2 sequence correctly spliced to the first C alpha exon. Analysis of the sequence revealed the presence of multiple stop codons in all three reading frames. The other 12 clones originated further upstream of the J alpha 11-2 exon and did not include the J alpha 11-2 exon, but rather arose from the joining of a cryptic splice donor signal to the usual TCR alpha C splice acceptor. This alternatively spliced transcript contained an open reading frame extending from the upstream J alpha 11-2 region to 82 nucleotides downstream of the beginning of the TCR C alpha region, and potentially encoded a 36 amino acid polypeptide. The remaining eight clones all contained the J alpha TA61 region correctly spliced to C alpha with two of these extending upstream of the J alpha TA61 exon. The predominance of J alpha 11-2-C alpha containing clones was confirmed by RNase protection assay using total RNA from kidney and spleen of scid mice. The 3' region of the transcript contained a fully conserved, correctly spliced TCR alpha C region which was polyadenylated at the 3' end. The truncated TCR alpha mRNA could be detected in preparations of cytoplasmic RNA, indicating that this transcript follows a normal RNA processing pathway. Our results demonstrate that the truncated TCR alpha mRNA expressed in normal mouse kidney is a germline J-C transcript resulting from transcription initiated predominantly upstream of the J alpha 11-2 region. This germline transcript in the kidney is undergoing alternative splicing leading to the appearance of an open reading frame coding for a short polypeptide. These results suggest that the product of this transcript may be functionally relevant.

Induction, suppression and superinduction of lymphokine mRNA in T lymphocytes.

The expression of several lymphokine gene is characterized by a common pattern of induction, suppression and superinduction. This pattern was studied at the level of cellular mRNA in the mouse T-lymphoma cell line EL4, the human T-leukemia line Jurkat and in normal human peripheral blood lymphocytes. Lymphokine mRNA was induced by stimulating the cells with the phorbol diester PMA (TPA), with or without T-lymphocyte mitogens. The induction of Interleukin-2, Interferon gamma and the Colony Stimulating Factor for granulocytes and macrophages was suppressed by Cyclosporin A at moderate concns. Furthermore, these mRNAs accumulated to extraordinarily high levels (superinduction) if the protein synthesis inhibitor cycloheximide was added during transcription. Superinduction was not due to an increased rate of transcription. CsA interrupted ongoing transcription of IL2 by a mechanism not dependent on the induction of a new protein. The co-ordinate regulation of these genes strongly suggests that common intracellular signals mediate their expression.