An immunodominant determinant for cytotoxic T lymphocytes (CTLs) exists in the hypervariable portion of human immunodeficiency virus-1 (HIV-1) gp160. Three mouse CTL lines (specific for isolates MN, RF, and IIIB) were examined for recognition of homologous determinants from distinct isolates. Only MN-elicited CTLs showed extensive interisolate cross-reactivity. Residue 325 played a critical role in specificity, with MN-elicited CTLs responding to peptides with an aromatic or cyclic residue and IIIB-induced cells recognizing peptides with an aliphatic residue at this position. CTL populations with broad specificities were generated by restimulation of IIIB-gp160 primed cells with MN-type peptides that have an aliphatic substitution at 325. This represents an approach to synthetic vaccines that can generate broadly cross-reactive CTLs capable of effector function against a wide range of HIV isolates.

Cancer immunosurveillance failure is largely attributed to the insufficient activation of tumor-specific class I major histocompatibility complex (MHC) molecule (MHC-I)-restricted CD8+ cytotoxic T lymphocytes (CTLs). DEC-205+ dendritic cells (DCs), having the ability to cross-present, can present captured tumor antigens on MHC-I alongside costimulatory molecules, inducing the priming and activation of tumor-specific CD8+ CTLs. It has been suggested that reduced levels of costimulatory molecules on DCs may be a cause of impaired CTL induction and that some tumors may induce the downregulation of costimulatory molecules on tolerogenic DCs. To examine such possibilities, we established two distinct types of murine hepatoma cell lines, named Hepa1-6-1 and Hepa1-6-2 (derived from Hepa1-6 cells), and confirmed that they display similar antigenicities, as well as identical surface expression of MHC-I. We found that Hepa1-6-1 had the ability to grow continuously after subcutaneous implantation into syngeneic C57BL/6 mice and did not prime CD8+ CTLs. In contrast, Hepa1-6-2 cells, which display reduced levels of adhesion molecules, such as Intercellular Adhesion Molecule 1 (ICAM-1), failed to grow in vivo and efficiently primed CTLs. Moreover, Hepa1-6-1-derived factors, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF) and α-fetoprotein (AFP), converted CD11chigh MHC-IIhigh DEC-205+ DC subsets into tolerogenic cells, displaying downregulated costimulatory molecules and having impaired cross-presenting capacities. These immunosuppressive tolerogenic DCs appeared to inhibit the induction of tumor-specific CD8+ CTLs and suppress their cytotoxic functions within the tumor. Together, the findings presented here provide a new method of cancer immunotherapy using the selective suppression, depletion or alteration of immunosuppressive tolerogenic DCs within tumors.

The major effector cells for cellular adaptive immunity are CD8(+) cytotoxic T lymphocytes (CTLs), which can recognize and kill virus-infected cells and tumor cells. Although CTLs exhibit strong cytolytic activity against target cells in vitro, a number of studies have demonstrated that their function is often impaired within tumors. Nevertheless, CTLs can regain their cytotoxic ability after escaping from the tumor environment, suggesting that the milieu created by tumors may affect the function of CTLs. As for the tumor environment, the patho-physiological situation present in vivo has been shown to differ from in vitro experimental conditions. In particular, low pH and hypoxia are the most important microenvironmental factors within growing tumors. In the present study, to determine the effect of these factors on CTL function in vivo, we examined the cytolytic activity of CTLs against their targets using murine CTL lines and the induction of these cells from memory cells under low pH or hypoxic conditions using antigen-primed spleen cells. The results indicated that both cytotoxic activity and the induction of functional CTLs were markedly inhibited under low pH. In contrast, in hypoxic conditions, although cytotoxic activity was almost unchanged, the induction of CTLs in vitro showed a slight enhancement, which was completely abrogated in low pH conditions. Therefore, antigen-specific CTL functions may be more vulnerable to low pH than to the oxygen concentration in vivo. The findings shown here provide new therapeutic approaches for controlling tumor growth by retaining CTL cytotoxicity through the maintenance of higher pH conditions.

The CDR3 regions of T cell receptor (TCR)-α and -β chains play central roles in the recognition of antigen (Ag)-MHC complex. TCR repertoire is created on the basis of Ag recognition specificity by CDR3s. To analyze the potential spectrum of TCR-α and -β to exhibit Ag specificity and generate TCR repertoire, we established hundreds of TCR transfectants bearing a single TCR-α or -β chain derived from a cytotoxic T cell (CTL) clone, RT-1, specific for HIVgp160 peptide, and randomly picked up TCR-β or -α chains. Surprisingly, one-third of such TCR-β containing random CDR3β from naive T cells of normal mice could reconstitute the antigen-reactive TCR coupling with RT-1 TCR-α. A similar dominant function of TCR-α in forming Ag-specific TCR, though low-frequency, was obtained for lymphocytic choriomeningitis virus–specific TCR. Subsequently, we generated TCR-α and/or -β transgenic (Tg) mice specific for HIVgp160 peptide, and analyzed the TCR repertoire of Ag-specific CTLs. Similar to the results from TCR reconstitution, TCR-α Tg generated CTLs with heterogeneous TCR-β, whereas TCR-β Tg-induced CTLs bearing a single TCR-α. These findings of Ag recognition with minimum involvement of CDR3β expand our understanding regarding the flexibility of the spectrum of TCR and suggest a predominant role of TCR-α chain in determining the preimmune repertoire of Ag-specific TCR.

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.

The effect of meal volume and fat content on gastroesophageal reflux was investigated in 20 asymptomatic healthy subjects. In each subject, intraesophageal pH monitoring was performed during a 3-hr postprandial period (PP) in the same position (supine or upright) on two successive days. On day 1, 500-ml low- and high-fat meals were ingested and, on day 2, an 800-ml low-fat meal was ingested. The acid exposure time was assessed as the percentage of time with a pH < 4.0. The acid exposure time in subjects in the upright position was significantly longer in the 800-ml group than in the 500-ml group for the entire PP (2.7 +/- 1.5%; mean +/- SE, 0.7 +/- 0.4%; P < 0.05). Of subjects in the supine position, the high-fat group showed significantly longer acid exposure time than the low-fat group both for the entire PP (7.6 +/- 3.0%, 0.7 +/- 0.5%; p < 0.05) and for the second hour (P < 0.05). We have demonstrated that differences in the meal volume and fat content influence gastroesophageal reflux in healthy asymptomatic subjects and that this influence varies with the position.

Based on the evidence that CD8+ cytotoxic T cells (CTL) precursors do not appear to distinguish between virus-infected cells and viral peptide-pulsed syngeneic cells, we have developed methods for priming class I MHC molecule restricted CD8+ CTL with such peptides without using any adjuvant. We were able to prime in vivo such CTL immunity lasting at least 6 months with a single i.v. injection of syngeneic 2200-3300 rad irradiated peptide-pulsed spleen cells, and even more efficiently with a very small number of irradiated class II MHC molecule expressing splenic dendritic cells (DC). No foreign serum source was necessary during the pulsing. Interestingly, we could not generate significant CTL activity with unirradiated or low dose (< 1100 rad) irradiated spleen cells. Because even purified DC required irradiation for optimal activity, because unirradiated B cells did not significantly inhibit the immunization with DC, and because B cell depletion did not substitute for irradiation, we believe that the effect of irradiation is more to determine homing of the cells than to eliminate interference by B cells. Intravenous immunization was much more effective than s.c. or i.p. immunization. CTL generated by this method could kill both peptide-pulsed syngeneic targets and targets endogenously expressing the whole gp160 gene. Moreover, we found that we could prime CD8+ CTL with the minimal 10-residue core peptide (RGPGRAFVTI) for optimal presentation by class I MHC molecules as efficiently as the original p18. These results suggested that DC bearing antigenic peptide may prime antigen-specific CD8+ CTL in vivo. These results offer useful information for development of synthetic peptide vaccines and immunotherapy.

In the case of viral infection, various viral proteins and genetic components are disseminated in the body. The former viral proteins may be captured by immature dendritic cells (DC) and the latter genetic components may stimulate the antigen-loading DC to maturate via specific Toll-like receptors (TLR), leading to the establishment of virus-specific cellular immunity; in particular, cytotoxic T lymphocytes (CTL) that control intracellular virions. Polyriboinosinic polyribocytidylic acid [poly(I:C)], which might reflect a natural genetic product from a variety of viruses during replication, has recently been identified as one of the critical stimuli for TLR3. Based on these observations, we speculated that stimulation of TLR3 with poly(I:C) might drive the direction of acquired/adaptive immunity to the cellular arm. Indeed, when BALB/c mice were immunized with purified recombinant HIV-1 envelope gp120 or influenza hemagglutinin (HA) protein together with poly(I:C), epitope-specific CD8(+) class I MHC molecule-restricted CTL were primed from naive CD8(+) T cells in vivo. In contrast, when the same proteins were immunized with lipopolysaccharide, a stimulant of TLR4, specific CTL were not primed at all. Moreover, we show here that immature DC could present processed antigen from captured purified protein in association with class I MHC molecules in the presence of poly(I:C), but not of LPS. These results indicate that we are able to manipulate the direction of acquired/adaptive effector immune responses using an appropriate stimuli and the findings presented in this paper will offer a new therapeutic strategy using poly(I:C) administration for priming antigen-specific CD8(+) CTL with purified viral protein in vivo.