Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy.

Adoptive cellular immunotherapy has proven to be a successful approach in preventing and curing cytomegalovirus infection and Epstein-Barr virus-associated lymphomas after bone marrow transplantation. Translation of this approach for preventing leukemia relapse after bone marrow transplantation might require ex vivo priming and long-term maintenance of leukemia blast-specific T cells. To accomplish this goal, procedures were optimized for the in vitro priming of naive CD8 using dendritic cells activated by CD40 ligation, interleukin-12 (IL-12), and IL-7. Using T lymphocytes and dendritic cells obtained from HLA-matched allogeneic bone marrow transplantation donors and leukemia blasts as a source of tumor antigens, anti-acute myeloid leukemia cytotoxic T lymphocytes (CTLs) were induced. In these experiments, it was found that though it is possible to induce CTLs using immature dendritic cells, IL-12, and IL-7, obtaining long-term CTLs requires the presence of CD4 T cells in the priming phase. Using this approach, long-term antileukemia CTL lines could be generated from 4 of 4 bone marrow donors. Because this procedure does not require definition of the target antigen and because it selects responding cells from a virgin T-cell repertoire, its general application is suggested in adoptive immunotherapy and in the definition of tumor rejection antigens.

Adoptive immunotherapy prevents prostate cancer in a transgenic animal model.

Cancer-related mortality can be decreased by prevention, early detection and improved therapies. Although animal models should be used to evaluate the success of cancer therapies, their usefulness is controversial. Many cancer therapies that have cured tumors in mice have not met with similar success when attempted in humans. Current animal models rely mainly on inoculating cell lines into animals, a method that does not reproduce the natural development of the tumor, both for the kinetics of induction and the anatomical site concerned. In this study, we have used an SV40 T-antigen-transgenic mouse model of prostate cancer in which the tumor spontaneously develops orthotopically with a disease progression that closely resembles the progression of human prostate cancer. We have used this model to test the suitability of adoptive cellular immunotherapy. Transfer of naive cells obtained from a T-antigen-negative congenic animal had significant but partial effects: it prevented development of malignant tumors, leaving just minor foci of residual tumor and/or hyperplasia. Adoptive transfer of memory lymphocytes specific for T-antigen, which is a prostatic self antigen in this model, prevented tumor development and progression without affecting the morphology and function of involved tissues. Treated animals were able to breed, and their survival was greatly increased. These results strongly suggest that adoptive immunotherapy should be successful in treating early stages of human prostate cancer.

Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: implications on immunogenicity and immunodominance.

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA-A*0201/Kb (A2/Kb) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA-A*0201-binding motif, we selected A2-binding peptides, immunized A2/Kb animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide-specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/Kb animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide-specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.

Immunodominance analysis of CTL responses to influenza PR8 virus reveals two new dominant and subdominant Kb-restricted epitopes.

In the present study, a systematic analysis of the influenza (Flu) PR8 determinants recognized by H-2b mice was undertaken. A single Db-restricted immunodominant epitope (NP(366)) was previously known in this system. Twenty-three different Flu PR8-derived peptides that bound either Kb or Db molecules in vitro were identified. Sixteen were immunogenic following peptide immunization of C57BL/6 mice, yet CTL induced by peptide immunization recognized PR8-infected target cells only in the case of the NP(366) and NS2(114) epitopes. Similarly, CTL responses following whole-PR8 virus immunization were detected only for the same two determinants. CTL recognizing these dominant epitopes had high avidity for peptide-pulsed target cells, with 5 to 200 pM of peptide required for 30% specific lysis. In contrast, most (80%) of the remaining epitopes were recognized with lower avidity (30% effective concentration in the range of 0.4-50 nM). Repeated in vitro stimulation of primary CTL cultures revealed one additional Kb-restricted epitope (M1(128)). This peptide bound Kb with high affinity (4.6 nM) and induced CTL that effectively recognized PR8-infected cells. These results suggest that 1) this epitope is produced by natural processing in relatively high amounts and 2) low precursor frequency might be related to the subdominant status of the M1(128) epitope. Taken together, these results illustrate the crucial contributions of MHC-binding capacity, and T cell repertoire availability, to the shaping of the repertoire of CTL specificities for Flu Ag virus.

Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans.

Our goal is to use peptide epitopes that are recognized by cytotoxic T lymphocytes (CTL) as immunogens for the development of prophylactic and therapeutic vaccines with chronic hepatitis B virus (HBV) infection being our first therapeutic target. Because most CTL peptide epitopes are poor immunogens, we specifically modified them by covalently attaching two additional components: a T helper peptide epitope and two lipid molecules. Using the murine influenza virus CTL epitope NP 147-155 as a model system, we found this construct to be highly immunogenic, and a single injection resulted in memory CTL induction that persisted for > 1 yr. Based on the animal studies, a vaccine was designed and tested for both safety and its ability to induce a primary CTL response in normal subjects. The three vaccine components included HBV core antigen peptide 18-27 as the CTL epitope, tetanus toxoid peptide 830-843 as the T helper peptide, and two palmitic acid molecules as the lipids. A dose escalation trial (5, 50, and 500 micrograms) carried out in 26 normal subjects showed that the vaccine was safe and able to induce a primary HBV-specific CTL response. A dose-response curve was observed and five out of five subjects responded to the 500-micrograms dose.

Random association between the peptide repertoire of A2.1 class I and several different DR class II molecules.

The interaction between synthetic peptides and A2.1 class I MHC molecules has been investigated using an inhibition of Ag presentation assay and unbiased peptide sets derived of either viral or eucaryotic origin. For the various sets, strong binding (defined as significant inhibition at the 30 micrograms/ml level) was detected in 7 to 46% of the peptides tested, with an overall frequency of 26%. A set of self-peptides derived from human beta 2 microglobulin was also included in the study. In this case, strong binding was detected in 3 of 15 peptides (20%), thus formally demonstrating a lack of self-/non-self-discrimination at the level of class I molecules. When the whole A2.1-binding database of 105 peptides thus generated was examined by sequence analysis, a significant correlation was found with a recently proposed A2.1-binding motif, whereas no particular positive or negative association was detected between the capacity to bind A2.1 and three different class II alleles (DR1, DR5, and DR7). Finally, using this approach, several peptides capable of binding both A2.1 and multiple DR alleles have been identified, suggesting possible candidates for development of peptide vaccines eliciting both class I and class II restricted responses.

Effects of dietary cellulose, pectin and oat bran on the small intestine in the rat.

The present study was undertaken to determine if three sources of dietary fiber would alter length, weight, DNA, protein or enzyme activity in the small intestine since various fibers are known to decrease intestinal absorption. Rats were fed semipurified diets that contained either 20% cellulose (C), 20% oat bran, 5% pectin (P) or no fiber source (FF). Leucine aminopeptidase activity were significantly greater in the P and C groups when compared with the FF group. There were no significant differences in sucrase activity. Animals in the P group had heavier and longer small intestine and heavier mucosa than the FF group. There were no significant differences in total mucosal DNA or protein. These results indicate that two sources of dietary fiber, cellulose and pectin, can change leucine aminopeptidase activity in the small intestine.