Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA.

The feasibility of dendritic cells (DC) for cancer immunotherapy after transfection by electroporation with mRNA encoding the human carcinoembryonic antigen (CEA) was investigated. Both, total RNA from the CEA(+) colon cancer cell line SW480 and mRNA transcribed in vitro from cDNA3.1-plasmids (pcDNA3.1+/-HisC) with a CEA-insert (ivt-CEA-mRNA, ivt-CEA/HisC-mRNA) were used. Labelled ivt-CEA-mRNA was detectable in DC by light and electron microscopy and by fluorescence-activated cell-sorting (FACS) even 15 min after electroporation. Four hours after transfection with ivt-CEA/HisC-mRNA, we detected specific expression of CEA and the histidine-tag by immunofluorescence microscopy and by FACS. CEA-specific T lymphocytes were successfully primed by transfected DC and were able to lyse CEA-expressing target cells, even from the CEA-expressing human colon adenocarcinoma cell line SW480. Thus, DC transfected by electroporation with CEA-mRNA are valuable tools for the immunotherapy of CEA(+) tumour entities.

A new mouse model for evaluating the immunotherapy of human colorectal cancer.

A new murine model of human colorectal cancer was generated by crossing human carcinoembryonic antigen (CEA) transgenic mice (H-2K(b)) with adenomatous polyposis coli (Apc1638N) knockout mice (H-2K(b)). The resulting hybrid mice developed gastrointestinal polyps in 6-8 months that progressed to invasive carcinomas with a similar pattern of dysplasia and CEA expression as observed in human colorectal cancer. These animals exhibited incomplete or partial tolerance to CEA as evidenced by delayed growth of CEA-expressing tumors and the inability to inhibit CEA-specific CTL responses. These results have important implications for understanding the role of CEA-specific immunity in human colon cancer patients and suggest that vaccine strategies targeting CEA may be feasible. This model provides a powerful system for evaluating antigen-specific tumor immunity against spontaneous tumors arising in an orthotopic location and permits evaluation of therapeutic vaccine strategies for human colorectal cancer.

Innovative treatments for pancreatic cancer.

Pancreatic cancer is the fifth leading cause of cancer deaths in the United States with little or no impact from conventional treatment options. Significant advances in understanding basic immunology have renewed interest in using immunotherapy to treat pancreatic cancer. Cancer immunotherapy, including humanized MAbs, cytokines, and potent vaccine strategies, has been successful in animal models and is being evaluated in clinical trials. Gene therapy is also being explored using methods to inactivate oncogenes, replace defective tumor suppressor genes, confer enhanced chemosensitivity to tumor cells, and increase immunogenicity of tumor cells. Angiogenesis, an essential step in the growth and metastasis of pancreatic cancer, has been targeted by many antiangiogenic agents. Several clinical trials have been initiated to evaluate the role of these innovative strategies in patients with pancreatic cancer with increasingly sophisticated correlative studies to learn more about the mechanisms of tumor rejection with these agents. The rapid translation of basic science discoveries to clinical trials should result in the development of new effective treatments for patients with pancreatic cancer.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes.

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule.

The generation of cytotoxic effector T cells requires delivery of two signals, one derived from a specific antigenic epitope and one from a costimulatory molecule. A phase I clinical trial was conducted with a non-replicating canarypoxvirus (ALVAC) constructed to express both human carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. This was the first study in cancer patients to determine if the delivery of costimulation with a tumor vaccine was feasible and improved immune responses. Three cohorts of six patients, each with advanced CEA-expressing adenocarcinomas, were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine (4.5 x 10(6), 4.5 x 10(7), and 4.5 x 10(8) plaque-forming units, PFU). Patients were vaccinated by intramuscular injection every 4 weeks for 3 months and monitored for side-effects, tumor growth and anti-CEA immune responses. ALVAC-CEA-B7.1 at doses up to 4.5 x 10(8) PFU was given without evidence of significant toxicity or autoimmune reactions. Three patients experienced clinically stable disease that correlated with increasing CEA-specific precursor T cells, as shown by in vitro interferon-gamma enzyme-linked immunoassay spot tests (ELISPOT). These three patients underwent repeated vaccination resulting in augmented CEA-specific T cell responses. This study represents the first use of costimulation to enhance antitumor vaccines in cancer patients. This approach resulted in CEA-specific immunity associated with stable diseases in three patients. This study also demonstrated that CEA-specific T cell responses could be sustained by repeated vaccinations. Although the number of patients was small, the addition of B7.1 to virus-based vaccines may improve immunological and stable diseases to vaccination against tumor-associated antigens with tolerable toxicity.

Strategies for cancer therapy using carcinoembryonic antigen vaccines.

Advances in molecular biology and immunology have renewed interest in the development of vaccines for the treatment or prevention of cancer. Research over the past 10 years has focused on the identification of suitable tumour antigens to use as targets for a variety of vaccine strategies. Carcinoembryonic antigen (CEA) was one of the first tumour antigens described, and is commonly expressed by a wide range of adenocarcinomas. Recent studies have identified several human-leukocyte-antigen-restricted epitopes (short peptides) within the CEA protein that can be recognised by human T lymphocytes (T cells). Although CEA-expressing tumour cells are generally weakly recognised by the immune system, several new strategies have been used to enhance immune responses against CEA. This includes using antibodies directed against CEA; inserting the CEA gene into recombinant viruses and bacteria as viral and bacterial vaccines; pulsing the CEA protein, peptides, DNA or RNA onto dendritic cells (specialised antigen-presenting cells); and combining CEA vaccines with cytokines or co-stimulatory molecules to increase vaccine effectiveness. Other factors that might be important in establishing systemic immunity against CEA are the dose, route, timing, and choice of vector and adjuvants for vaccine administration. Further research in understanding the fundamental processes involved in tumour-cell recognition by the immune system, better animal models, and improved clinical trial designs will help to define the full potential of CEA as a target for cancer vaccine development.

Binding of longer peptides to the H-2Kb heterodimer is restricted to peptides extended at their C terminus: refinement of the inherent MHC class I peptide binding criteria.

MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated.

Point mutations in the beta chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area.

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.

An in vitro study of the dynamic features of the major histocompatibility complex class I complex relevant to its role as a versatile peptide-receptive molecule.

The major histocompatibility complex class I complex consists of a heavy chain and a light chain (beta2-microglobulin, beta2m), which assemble with a short endogenously derived peptide in the endoplasmic reticulum. The class I peptide can be directly exchanged, either at the cell surface or, as recently described, in vesicles of the endocytic compartments, thus allowing exogenous peptides to enter the class I presentation pathway. To probe the interactions between the components of the class I molecule, we analyzed the exchange of peptide and beta2m by using purified, recombinant H2-Kb/peptide complexes in a cell-free in vitro system. The exchange of competitor peptide was primarily dependent on the off-rate of the original peptide in the class I binding groove. Peptide exchange was not enhanced by the presence of exogenous beta2m, as exchange occurred to the same extent in its absence. Thus, the exchange of peptide and beta2m are independent events. The exchange rate of beta2m also was not affected by the dissociation rates of the original peptides. Furthermore, peptides could substantially exchange into class I molecules over a pH range of 5.5 to 7.5, conditions prevalent in certain endocytic compartments. We conclude that the dynamic properties of the components of class I molecules explain its function as a highly peptide-receptive molecule. The major histocompatibility complex class I can readily receive peptides independent of the presence of exogenous beta2m, even at a low pH. Such properties are relevant to class I peptide acquisition, which can occur at the cell surface, as well as in specialized endosomes.

Role of glutamine in the immune response in critical illness.

The dependence of human lymphocyte functions on the exogenous provision of glutamine (GLN) was evaluated in a series of in vitro experiments. The transcription of early activation markers (IL-2, IL-2-receptor, IL-4, IL-4, GM-CSF, IFN gamma) as evaluated by polymerase chain reaction was observed even in the absence of exogenous GLN. In contrast, later events of lymphocyte activation including cytokine production, proliferation of lymphocytes and lymphokine-activated killer cell activity were found to depend on exogenous GLN provision. These in vitro results are discussed in the context of established data on the reduction of peripheral blood GLN concentrations in critically ill patients and in view of recent studies reporting improved outcome of critically ill patients following GLN substitution. By and large, these data support the concept of GLN substitution in critical illness. However, the definition of indications and dose-response relationships clearly require further clinical studies.

In vivo CTL immunity can be elicited by in vitro reconstituted MHC/peptide complex.

The use of peptides as a vaccine is a potentially powerful immunization strategy. We explored the possibility of inducing an efficient cytotoxic T lymphocyte (CTL) mediated immune response in mice, using in vitro reconstituted major histocompatibility complex (MHC) class I/peptide complexes as the immunogen. Recombinant derived H-2Kb and beta 2-microglobulin (beta 2m) were properly folded into an MHC class I complex using the vesicular stomatitis virus (VSV)-8mer from the natural nucleocapsid proteinN52-59 (RGYVYQGL), an immunodominant Kb epitope in C57BL/6 (B6) mice. After immunizing mice with the H-2Kb class I/VSV peptide complex and a subsequent in vitro stimulation with the VSV peptide alone, a specific CTL response was demonstrated. The method was also applicable to other peptides, for example, the Sendai virus (SV) peptideN324-332 (FAPGNYPAL). The CTL response was mediated by CD3+/CD8+ T cells and was shown to be allele specific, as only peptide loaded target cells expressing the H-2Kb allele could be recognized. It is of interest that extremely small amounts of injected MHC class I/peptide complex (i.e. 500 pg) could generate a measurable CTL response. The MHC class I/peptide complex had to be intact and properly folded to elicit an immune response, suggesting that the complex protected the peptide for internalization by antigen presenting cells (APCs) or for delivering to the proper site for peptide exchange on the cell surface of APCs. The described immunizing method can be routinely used to prime a CTL response by employing in vitro folded MHC class I/peptide complexes, without the use of adjuvants. It appears to be efficient, sensitive and specific. By using the recombinant protein system, unlimited amounts of MHC class I/peptide complex can be produced for immunization. Moreover, this protocol permits different in vitro combinations of allelic MHC class I molecules and peptide variants.

Nitric oxide-independent inhibitory effects of L-arginine analog NG-monomethy-L-arginine on the generation of interleukin-2 activated cytotoxic activity in humans.

Nitric oxide (NO) derived intracellularly from L-arginine (Arg) is indispensable for optimalgeneration of lymphokine-activated killer (LAK) cell activity in rodents. Still unclear, however, is its role in humans. To address this question human peripheral blood mononuclear cells (PBMC) from healthy donors were cultured in L-arginine free medium supplemented with recombinant interleukin-2 (rIL-2) and in the presence of exogenous L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of the NO synthetic pathway. Cultured PBMC were tested for cytotoxic activity, proliferative capacity, and expression of phenotypic and activation markers (CD3, CD4, CD8, CD16, CD56 and CD25). Culture supernatants were assayed for nitrite (NO2-) and tumor necrosis factor-alpha (TNF-alpha) production. We found that NMMA inhibits the generation of optimal LAK cell activity when no exogenous Arg is supplied. Similar effects were also observed on proliferation, expression of IL-2 receptor induced upon rIL-2 stimulation and on TNF-alpha production. Sodium nitroprusside (SNP), used as a source of exogenous NO could not overcome this effect of NMMA on LAK cell activity. NO2- production was virtually undetectable in culture supernatants. Thus, NMMA affects in an NO-independent manner rlL-2 induced LAK activity in human PBMC.

Tyrosine kinase-dependent and -independent events induced by interleukin-2 stimulation: interleukin-2-mediated NO production required for the induction of lymphokine-activated killer cell activity in rat splenocytes is tyrosine kinase independent.

Nitric oxide (NO) has recently been shown to be an indispensable co-factor in the generation of lymphokine-activated killer (LAK) cells induced by interleukin-2 (IL-2). Upon stimulation with IL-2, cells endowed with specific receptors undergo phosphorylation of substrates mediated by protein tyrosine kinases (PTK). In this work we utilized a well-characterized PTK inhibitor, genistein (GEN), to address the role of PTK on NO-dependent LAK cell generation. The effects of GEN were tested on the expression of the inducible NO synthase (iNOS) gene, proliferation, generation of cytotoxic activity and production of NO upon IL-2 stimulation of rat splenocytes. We report here that GEN displays profound inhibitory effects on recombinant (r)IL-2 induced proliferation and on LAK cell generation, while only marginally affecting NO production, measured as NO2-. In contrast, a specific inhibitor of the NO synthetic pathway (NG-monomethyl-L-arginine; NMMA) blocked generation of LAK cells and NO production without affecting cell proliferation. If added directly to the cytotoxicity tests, GEN exerted minor inhibitory effects, not exceeding 25% of control tests, while NMMA was completely ineffective. Sodium nitroprusside (SNP), a non-enzymatic NO-releasing substance, restored LAK cell generation in cultures performed in the presence of NMMA, but not in those performed in the presence of GEN. These results indicate that IL-2-induced NO production is a PTK-independent event. IL-2-stimulated LAK cell generation obligatorily requires the concurrent activation of PTK dependent and independent signal transduction pathways.

Generation of lymphokine-activated killer activity in rodents but not in humans is nitric oxide dependent.

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG-monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO2-), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Glutamine requirements in the generation of lymphokine-activated killer cells.

The role of glutamine (GLN) in the generation of lymphokine-activated killer (LAK) cell activity was investigated. LAK cells were derived from healthy donors and peripheral blood mononuclear cells (PBMC) were obtained using either unseparated PMBC or DR(-) CD3(-) CD16(+) CD56(+) enriched cells. PBMC were cultured for 6 or 10 days in medium supplemented with recombinant interleukin-2 (rlL-2; 100 U/ml) in the presence of different concentrations of GLN. K562 (natural killer-NK-sensitive targets), 1301 and U-937 (NK-resistant targets) cells were used as targets in the cytotoxic assays. Furthermore, the limiting dilution (LD) culture system was applied as an alternative to the bulk cell culture system. It was found that GLN affects the lytic potential of cultured cells while the frequency of responding cells did not significantly differ between the compared cell cultures performed in the presence of different amounts of GLN. Data on cell proliferation with IL-2 stimulation showed significant differences in cultures performed in the presence or absence of GLN. The results of present investigation suggest a supportive role of GLN in the generation of LAK cells. GLN deficit affects LAK cell killing activity by limiting the number of generated effector cells while acquisition of broad-range killing capacity was not affected.

Exogenous glutamine requirement is confined to late events of T cell activation.

Glutamine is required for the proliferation of lymphocytes, but quantitative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM-1 mM) on the in vitro response of human peripheral blood mononuclear cells (PBMC) to a mitogenic anti-CD3 monoclonal antibody (mAb) was investigated. Expression of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequentially analyzed. Proliferation was measured by a 3H-thymidine incorporation assay. mRNA coding for IL-2, IL-2 receptor, IL-4, IL-5, GM-CSF, and IFN-gamma was detectable independently from exogenous glutamine provision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD71 as well as the production of IFN-gamma were found to require exogenous glutamine supply. In contrast, production of TNF-alpha could be observed in the absence of glutamine and was increased to a limited extent by exogenous glutamine. The overall lymphocyte response as reflected by entering into the cell cycle and proliferation was directly correlated with the glutamine concentration of the culture medium. Efficient progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM to 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti-CD3 stimulation of a CD4+ T cell clone.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine peptide-supplemented long-term total parenteral nutrition: effects on intracellular and extracellular amino acid patterns, nitrogen economy, and tissue morphology in growing rats.

Glutamine (GLN) is a nonessential amino acid that is not included in current regimens for parenteral nutrition because of its chemical instability. This study tested the hypothesis that GLN supplementation during long-term total parenteral nutrition (TPN) (3 weeks) would enhance GLN availability, thereby improving nitrogen economy and growth in a growing rat model: Standard TPN delivering 300 kcal/kg per day (lipid:carbohydrate = 1.1) including 2.1 g of nitrogen per kilogram per day in an all-in-one solution was compared with an isonitrogenous, isocaloric, and isovolemic TPN regimen with 0.29 g of nitrogen per kilogram per day substituted by GLN derived from the dipeptides glycyl-GLN and alanyl-GLN (TPN GLN). Enterally fed controls were included. Analysis was confined to nonbacteremic animals with negative blood culture, in which extracellular and intracellular amino acid concentrations including GLN, nitrogen balance, serum protein concentrations, growth, and histologic sections of liver and small-bowel mucosa (light and scanning electron microscopy) were evaluated. Hepatic intracellular GLN concentrations were significantly lower, in animals receiving GLN-free TPN (11.7 +/- 1.6 nmol/mg fat-free dry and solid tissue mass, n = 9) compared with both GLN-supplemented TPN (16.0 +/- 3.0, n = 7) and enteral feeding (18.2 +/- 1.8, n = 6) (p < .001). Corresponding results were found for intracellular GLN concentrations in skeletal muscle (TPN standard 12.5 +/- 3.1, TPN GLN 14.7 +/- 3.1, enteral control 17.3 +/- 2.3, p < .05), intestinal mucosa, and spleen as well as for plasma concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)