Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines.

Synthetic peptide analogues of sequences in the HER-2 protooncogene (HER-2) were selected based on the presence of HLA-A2.1 anchor motifs to identify the epitopes on HER-2 recognized by ovarian tumor-reactive CTL. 19 synthetic peptides were evaluated for recognition by four HLA-A2 ovarian-specific cytotoxic T lymphocyte (CTL) lines obtained from leukocytes associated with ovarian tumors. The nonapeptide E75 (HER-2, 369-377:KIFGSLAFL) was efficient in sensitizing T2 cells for lysis by all four CTL lines. This peptide was specifically recognized by cloned CD8+ CTL isolated from one of the ovarian-specific CTL lines. E75-pulsed T2 cells inhibited lysis by the same CTL clone of both an HLA-A2+ HER-2high ovarian tumor and a HER-2high cloned ovarian tumor line transfected with HLA-A2, suggesting that this or a structurally similar epitope may be specifically recognized by these CTL on ovarian tumors. Several other HER-2 peptides were recognized preferentially by one or two CTL lines, suggesting that both common and private HER-2 epitopes may be immunogenic in patients with ovarian tumors. Since HER-2 is a self-antigen, these peptides may be useful for understanding mechanisms of tumor recognition by T cells, immunological tolerance to tumor, and structural characterization of tumor antigens.

An improved method to estimate ultrasonic absorption in agar-based gel phantom using thermocouples and MR thermometry.

In this paper, a novel experimental set-up was developed that measures the absorption coefficient. The proposed system was evaluated in an agar-based gel phantom. The new experimental system provides accurate and fast measurement of the rate of temperature change within the phantom. The rate of temperature change was measured using thermocouple and was confirmed using MR thermometry. An ultrasonic transducer with a broad beam was used in order to reduce the conduction effect. The absorption coefficient of the agar-based phantom was 0.26 dB/cm-MHz using 4% agar, 30% evaporated milk and 4% silica. The absorption coefficient increased by increasing the volume of the evaporated milk, and agar. The absorption coefficient increased at low silica concentration (<4%) and then decreased at higher concentration of silica (>4%). By proper selection of evaporated milk, agar and silica concentration, it is possible to achieve similar coefficient like in soft tissues. Acoustic absorption measurement is considered as a difficult measurement in ultrasonics because obtaining the precise temperature change in the focus is challenging. Due to the quick and accurate placement of the thermocouple at the ultrasonic beam, it is possible with the proposed system to perform absorption measurement is less than one minute.

Tissue differences in the modulation of rat cytochromes P450 and phase II conjugation systems by dietary doses of phenethyl isothiocyanate.

Rats were fed diets supplemented with phenethyl isothiocyanate (PEITC) at 0.06 (low dose, dietary intake level), 0.6 (medium dose) and 6.0 micromole/g (high dose), and xenobiotic-metabolising enzymes were monitored in liver, lung and kidney. At the low dose, inhibition of the hepatic O-dealkylation of ethoxy- and methoxyresorufin was noted, whereas at the high dose increases in the O-depentylation of pentoxyresorufin and O-debenzylation of benzyloxyquinoline were observed, whereas p-nitrophenol hydroxylase was inhibited. Hepatic bioactivation of 2-amino-3-methylimidazo-[4,5-f]quinoline to mutagens was not influenced by the PEITC-treatment. In the lung, at the high dose, ethoxyresorufin dealkylation was elevated and that of pentoxyresorufin suppressed; no significant changes were seen in the kidney. Quinone reductase was markedly elevated at all doses in liver, but the lung enzyme was refractive whereas in the kidney a modest rise was observed at the high dose. Hepatic glutathione S-transferase activity was stimulated by PEITC-treatment, but no effect was evident in the lung or kidney. It is concluded that the effects of PEITC on xenobiotic-metabolising systems are dose- and tissue-dependent, with the liver being the most sensitive and the lung generally resistant. Increased detoxication rather than cytochrome P450 inhibition is the likely mechanism of the chemopreventive activity of PEITC.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model.

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3(+)CD8(+) but not the CD3(+)CD4(+) cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Increased sensitivity of adriamycin-selected tumor lines to CTL-mediated lysis results in enhanced drug sensitivity.

The emergence of drug resistance to chemotherapeutic agents is a major cause of treatment failure in cancer therapy. Therefore, much effort has been aimed at circumventing or reversing this undesired effect. Recently, we found that tumor cell lines selected for their multidrug-resistant phenotype can also exhibit increased levels of TAP mRNA and MHC class I proteins. This raised the question of whether drug-resistant tumors are more readily recognized by MHC-restricted CTLs. In this report, we show that five of five MHC class I+ tumor cell lines grown in medium containing Adriamycin developed into variants that expressed higher levels of MHC class I than did their corresponding parental cell lines. This was not observed with a MHC class I- cell line. No similar association was noted for changes in the expression of either HER-2 or intercellular adhesion molecule 1 protein. We also found that MHC class I+ drug-selected variants were more readily lysed by MHC-restricted, tumor-associated CTLs than were the drug-sensitive parental cell lines. When the drug-selected variants were cocultured with the same CTLs to eliminate tumor cells expressing higher levels of MHC-I (MHC-Ihi), the CTL-resistant tumor cells exhibited a drug sensitivity profile similar to that of the parental cell lines that were not exposed to Adriamycin. These findings suggest that certain chemotherapeutic drugs may increase the immunogenicity of some tumors, and that CTL immunotherapy may help reverse drug resistance.

Inhibition of the mutagenicity of aromatic amines by the plant flavonoid (+)-catechin.

Addition of the plant phenolic flavonoid (+)-catechin to rat liver microsomes inhibited the mutagenicity of the aromatic amines 2-aminofluorene and 4-aminobiphenyl in the Ames test. Similarly, (+)-catechin decreased the mutagenicity of N-hydroxy-4-amino-biphenyl, the proximate carcinogen, but, in contrast, had no effect on the mutagenicity of other direct-acting carcinogens such as N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine. In vitro addition of (+)-catechin gave rise to a dose-dependent inhibition of the cytochrome P-450-dependent benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities. This was achieved by impairment of the electron flow from the reduced pyridine nucleotide to the cytochrome. However, administration of (+)-catechin to rats had no effect on the in vitro mixed-function oxidase activities. It is concluded that the (+)-catechin inhibits the mutagenicity of aromatic amines in the Ames test by interfering with their cytochrome P-450-dependent bioactivation and by direct interaction with the proximate carcinogen, but the former mechanism is unlikely to occur in vivo because the high doses of the flavonoid required are not achieved.

Tumor cytolysis by lymphocytes infiltrating ovarian malignant ascites.

Tumor-associated lymphocytes (TAL) were isolated from the ascitic fluid of patients with adenocarcinoma of the ovary. These cells proliferated and expanded by 100-600-fold as either CD3+ CD4+ or CD3+ CD8+ cultures in the presence of moderate concentrations (50-200 cetus units/ml) of recombinant interleukin 2 and reached high numbers (5 x 10(8)-1 x 10(9)). After expansion of 16 TAL samples from 15 patients, 5 of the 7 isolated ovarian cytotoxic T-lymphocyte cell lines of T-cell receptor (TCR) (alpha beta)+ CD3+ CD8+ CD4- phenotype exhibited preferential cytolytic activity against autologous tumor targets and significantly lower cytolytic activity against allogeneic tumor targets and the natural killer-sensitive cell line K562. The cytolytic activity of the CD8+ TAL was inhibited by operationally anti-TCR (alpha beta) monoclonal antibody and monoclonal antibody specific for the CD3 differentiation antigen, indicating that the TCR and CD3 are involved in the cytolytic process. The other TAL cultures demonstrated similar cytolytic activity against both autologous and allogeneic tumors. The phenotype of these TAL was predominantly TCR (alpha beta)+ CD3+ CD4+ CD8-. Certain CD3+ CD8+ T-cell clones isolated from representative TAL exhibited preferential autologous tumor-specific cytotoxicity that may be major histocompatibility complex restricted. Other CD3+ CD8+ and CD3+ CD4+ clones exhibited nonmajor histocompatibility complex restricted cytotoxicity. These results demonstrate that CD3+ CD4+ and CD3+ CD8+ T-cells present in the ovarian malignant ascites can be propagated in large numbers and for long time intervals as T-cell lines in vitro. This finding may be significant for further investigation of ovarian tumor-specific cytotoxic T-lymphocytes and future adoptive specific immunotherapy studies.

Polyadenylic acid polymerase activity in normal and leukemic human leukocytes.

Soluble polyadenylic acid [poly(A)] polymerase and poly(A) nucleases content of normal human blood lymphocytes and leukemic blood cell populations was determined. Blood lymphocytes from seven normal individuals were used as controls. Leukemic cells were obtained from 69 patients with various types of acute and chronic leukemias. Chronic lymphocytic leukemias presented poly(A) polymerase values with a mean of 9 +/- 4 (S.D.). Although most of the chronic lymphocytic leukemia cases presented poly(A) polymerase activities similar to those of normal lymphocytes (3 +/- 3), a small number fell into the specific activity values of acute leukemias, which were significantly higher and covered a wider range. The mean values for acute myeloblastic, acute monoblastic, and acute lymphoblastic leukemias were 53 +/- 50, 21 +/- 8, and 29 +/- 14, respectively. A statistically significant difference was found between chronic and acute leukemias (p less than 0.01). The observed differences in poly(A) polymerase levels of acute lymphoblastic leukemia versus chronic lymphocytic leukemia persisted after fractionation of the crude extracts and, furthermore, they could not be attributed to differences in the levels of poly(A)-degrading enzymes [poly(A) endo- and exonucleases]. Fractionation of leukemic extracts on Sephadex G-75 revealed two molecular forms of poly(A) polymerase activity.

Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxic T lymphocytes.

Identification of naturally processed peptides recognized by tumorspecific CTLs may lead to epitope-specific tumor vaccines. Because these epitopes may be expressed differently on epithelial tumors and may differ in their ability to induce CTL in vivo, we have isolated the HLA-A2-peptide complexes by immunoaffinity from an established ovarian tumor line transfected with and expressing HLA-A2 gene. High-performance liquid chromatography-fractionated peptides were used to reconstitute epitopes recognized on HLA-A2 by three HLA-A2+ CD8+ CTL lines. These lines recognized at least three of the same groups of fractions (designated SKOV3.A, -B, and -C) but showed differences in the pattern of recognition of other fractions. To gain insight in the epitope distribution by freshly isolated ovarian tumors, we compared the recognition of peaks SKOV3.B and -C with the corresponding peaks from an ovarian tumor (OVA-6) that expressed similar levels of HLA-A2, using one of these lines (CTL-OVA-5) as indicator. CTL-OVA-5 recognized a large number of epitopes from peaks B and C rechromatographed on more resolving high-performance liquid chromatography gradient. Although a number of peaks appeared to be coincident on both SKOV3 and OVA-6, an even higher number appeared either not to overlap or to overlap only partially. These findings, which represent the first analysis of the epitopes presented by a patient tumor, suggest that the use of tumor line-derived peptides for vaccination may require selection of the epitopes corresponding to the ones presented by freshly isolated human tumors.

Role of ketone bodies in the diabetes-induced changes in hepatic mixed-function oxidase activities.

In order to assess the role of ketone bodies in the diabetes-induced changes in hepatic mixed-function oxidase activity, rats rendered hyperketonaemic by dietary administration of medium chain triacylglycerols were compared with streptozotocin treated rats. Both groups of animals became hyperketonaemic but only the latter were hyperglycaemic. Treatment with streptozotocin or medium chain triacylglycerols gave rise to marked increases in the O-dealkylations of ethoxyresorufin, ethoxycoumarin and pentoxyresorufin, the p-hydroxylation of aniline and the N-demethylation of dimethylnitrosamine. It is concluded that the streptozotocin-induced changes in hepatic mixed-function oxidases are mediated, at least partly, by the high levels of ketone bodies.

Anthraflavic acid is a potent and specific inhibitor of cytochrome P-448 activity.

Consideration of the computer-optimised dimensions of anthraflavic acid indicates that it is essentially a planar molecule with a large area/depth ratio, that would preferentially interact with the polycyclic aromatic hydrocarbon-induced family of cytochrome P-450 proteins (cytochromes P-448). Anthraflavic acid was a potent inhibitor of the O-deethylations of ethoxycoumarin and ethoxyresorufin, both catalysed primarily by cytochromes P-448, in Arochlor-1254-induced hepatic microsomes. Similarly anthraflavic acid markedly inhibited the mutagenicity of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-I) in the Ames test. In contrast, it has no effect on the dealkylation of pentoxyresorufin, a reaction catalysed primarily by the phenobarbital-induced cytochromes P-450, and NADPH-dependent reduction of cytochrome c. It is concluded that anthraflavic acid is a potent and specific inhibitor of cytochrome P-448 activity.

Induction of hepatic CYP1A2 by the oral administration of caffeine to rats: lack of association with the Ah locus.

Caffeine was administered to male Wistar albino rats for two weeks at three concentrations, namely 0.1, 0.2 and 0.3%, and hepatic cytochrome P450-dependent mixed-function oxidase determined. Caffeine administration gave rise to a marked, dose-dependent increase in the O-deethylation of ethoxyresorufin and, to a lesser extent, in the O-depentylation of pentoxyresorufin. Erythromycin N-demethylase, p-nitrophenol hydroxylase and lauric acid hydroxylase activities, as well as total cytochrome P450 content were unaffected by this treatment. Immunoblot analysis revealed that caffeine gave rise to a dose-dependent increase in the hepatic CYP1A2, and at the highest dose only, CYP2B apoprotein levels. Apoprotein levels of CYP3A and CYP2E1 were not modulated by the treatment with caffeine at all dose levels studied. Caffeine could not displace [3H]TCDD from the rat hepatic cytosolic Ah receptor. Computer analysis showed that caffeine is essentially a planar molecule with an area/depth ratio 4.8, characteristic of CYP1A substrates/inducers. Molecular modelling revealed that the caffeine molecule could orientate itself within the putative CYP1A2 active site so as to facilitate demethylation of the N-1, N-3 and N-7 positions. However, at physiological pH, the N-9 nitrogen atom is likely to be partially protonated, allowing it to participate in an electrostatic interaction with the negatively-charged glutamate 318-residue, favouring N-3 demethylation, the major pathway of metabolism in both humans and animals. In conclusion caffeine, being essentially planar, is an inducer of CYP1A2 in rat liver.

Peptide priming of cytolytic activity to HER-2 epitope 369-377 in healthy individuals.

The presence of tumor-reactive CTLs in tumor infiltrates and in the peripheral blood of cancer patients demonstrates an immune response against tumors that apparently cannot control disease spread. This raises concerns as to whether amplification of this response may be useful during disease progression. Induction of tumor-reactive CTLs in healthy donors at risk, as well as in patients free of disease, may be therapeutically important, based on the hypothesis that CTLs that recognize tumors early may be more effective in containing their progression than CTLs that expand only when the disease progresses. To address the feasibility of priming cytolytic activity in healthy donors, we used the HER-2 peptide E75 (369-377) as an immunogen and autologous peripheral blood mononuclear cell-derived dendritic cells as antigen-presenting cells. We found that of 10 healthy donors tested, two responded at priming with E75 presented on autologous dendritic cells by induction of E75-specific CTL activity. Three other responders were identified after two additional restimulations. Of these five responders, three recognized E75 presented on the ovarian tumor line SKOV3.A2, as demonstrated by cold-target inhibition experiments. Induction of cytolytic activity at priming was enhanced in responders by tumor necrosis factor-alpha and interleukin 12 but not in the nonresponders. AlphaB7.1 monoclonal antibody added at priming enhanced induction of lytic activity in only one of the four nonresponding donors tested, suggesting that in the majority of donors, E75-precursor CTLs were not tolerized. Because of the possibility that disease may develop in nonresponders, strategies to improve the immunogenicity of tumor antigens for healthy donors may be required for development of cancer vaccines.

Enhancement of cell-mediated immunity in melanoma patients immunized with murine anti-idiotypic monoclonal antibodies (MELIMMUNE) that mimic the high molecular weight proteoglycan antigen.

The purpose of this study was to determine whether a combination of two anti-idiotypic antibodies that mimic the high molecular weight proteoglycan antigen found on most melanoma tumors was capable of enhancing cellular immunity in vaccinated high-risk patients with melanoma. Twenty-eight stage I-IV high-risk patients with melanoma were immunized with a mixture of variable concentrations of MELIMMUNE-1 and MELIMMUNE-2, along with the adjuvant SAF-m, using two immunization schedules. Peripheral blood mononuclear cells were collected before the first immunization and 4 weeks after the final immunization and tested for in vitro proliferation to MELIMMUNE-1 and MELIMMUNE-2 and for cytotoxicity against 51Cr-labeled target cell lines. Additionally, supernatants from in vitro proliferation cultures were tested for interleukin 10 and IFN-gamma levels. Significant in vitro proliferation to MELIMMUNE-1 and MELIMMUNE-2 were observed in postimmunization samples but not in prevaccination samples. The mean stimulation index for MELIMMUNE-2 (33.7 +/- 0.6) was significantly higher than that for MELIMMUNE-1 (13.9 +/- 0.3; P < 0.025). Supernatants obtained from 78% of the in vitro stimulated cultures pre- or postvaccination contained significant levels of interleukin 10 (range, 0.43-142 pg/ml), whereas IFN-gamma levels were elevated in 53% of postvaccination samples (range, 3-245 pg/ml) but not prevaccination samples. More importantly, we were able to generate specific CTL responses in 43% of the patients, which correlated with elevated IFN-gamma levels. These results indicate that MELIMMUNE enhances cell-mediated immunity in patients with melanoma.

Vaccine implications of folate binding protein, a novel cytotoxic T lymphocyte-recognized antigen system in epithelial cancers.

The immune system can be efficiently stimulated and targeted to specific antigens expressed exclusively or preferentially by experimental cancers. The foremost limitations to extending this vaccine technology to the prevalent epithelial-derived cancers are the lack of: (a) identified tumor-associated antigens recognized by cellular immunity; (b) antigens expressed on the majority of tumor cells during disease progression; and (c) immunogenic CTL epitopes. To date, only HER-2/neu has been shown to be the source of naturally occurring, MHC-restricted, CTL-recognized peptides in epithelial tumors. In this study, we demonstrate that the human high-affinity folate binding protein (FBP), which is a source of antigenic peptides recognized in ovarian cancer, is also recognized in breast cancer. Both immunodominant E39 (FBP, 191-199) and subdominant E41 (FBP, 245-253) epitopes are presented by HLA-A2 in these cancers. These peptides are efficient at amplifying the response of tumor-associated lymphocyte populations in terms of lytic function, enhanced proliferation, and specific IFN-gamma release. On a per cell basis, tumor-associated lymphocytes stimulated with the FBP peptides exhibit enhanced cytotoxicity not only against peptide-loaded targets but also against FBP-expressing epithelial tumors of different histologies. Furthermore, FBP peptides induced E39-specific CTLs and E39- and E41-specific IFN-gamma and IP-10 secretion in certain healthy donors. The broad distribution of FBP among >90% of ovarian and endometrial carcinomas, as well as 20-50% of breast, lung, colorectal, and renal cell carcinomas, along with pronounced differential overexpression in malignant tissues compared with the extremely limited expression in normal epithelium, suggests the exciting potential of a widely applicable FBP-based vaccine in epithelial cancers.

Proliferative and cytokine responses to class II HER-2/neu-associated peptides in breast cancer patients.

Previous studies have characterized the reactivity of CD8+ CTLs with ovarian and breast cancer. There is little information about the antigens and epitopes recognized by CD4+ T cells in these patients. In this study, we analyzed the ability of T cells from peripheral blood mononuclear cells of breast cancer patients to recognize HER-2/neu (HER-2) peptides. We found that 13 of 18 patients responded by proliferation to at least one of the HER-2 peptides tested. Of these peptides, one designated G89 (HER-2: 777-789) was recognized by T cells from 10 patients. Seven of nine responding patients were HLA-DR4+, suggesting that this peptide is recognized preferentially in association with HLA-DR4. Analysis of the specificity and restriction of the cytokine responses to G89 by G89-stimulated T cells revealed that these cells secreted significantly higher levels of IFN-gamma than interleukin 4 and interleukin 10, suggesting priming for a Th0-T helper 1 response. The same pattern of cytokine responses was observed to the intracellular domain of HER-2 protein, suggesting that G89-stimulated T cells recognized epitopes of the HER-2 protein in association with HLA-DR4. Because HLA-DR4 is present in 25% of humans, characterization of MHC class II-restricted epitopes inducing Th0-T helper 1 responses may provide a basis for the development of multivalent HER-2-based vaccines against breast and ovarian cancer.

Ovarian and breast cytotoxic T lymphocytes can recognize peptides from the amino enhancer of split protein of the Notch complex.

In this study we investigated recognition by ovarian tumor associated lymphocyte (OVTAL), and breast tumor associated lymphocytes (BRTAL), of peptides corresponding to the sequence 125-135 of the Aminoenhancer of split (AES) protein. Three of these peptides designated as G75:AES1/2 (128-135), G60: AES1/2 (127-137) and G61: AES1/2 (125-133) correspond to the wildtype AES sequence, while the fourth G76:GPLTPLPV, AES1/2 (128-135) corresponds to a variant sequence of the peptide G75 with the N-terminal Leu substituted to glycine. These sequences were chosen for study because mass-spectrometric analysis (MS) of a CTL active HPLC peptide fraction eluted from immunoaffinity precipitated HLA-A2 molecule, revealed: (a) the presence of an ion with a mass-to-charge ratio (m/z) of 793 which was more abundant than other ions of similar masses; (b) the tentatively reconstituted sequence of the ion 793 matched the sequence of peptide G76. We found that AES peptides G75 (128-135) and G76 (128-135) (L128G) reconstituted CTL recognition at concentrations ranging between 200-500 nM. These concentrations are lower than concentrations reported to activate effector function of CTL recognizing other epithelial tumor Ag. Furthermore, analysis with cloned CD8+ T cells indicated that G75 and G76 were not cross-reactive specificities, suggesting a key role for the N-terminal residues of the variant peptide in dictating specificities. Since the AES proteins are part of a set of transcriptional repressors encoded by the Enhancer of split [E(spl)] genes, and since these repressors are activated to suppress cell differentiation in response to Notch receptors signalling, the AES peptides may represent a novel class of self-antigens that deserve further consideration as tumor Ag in epithelial cancers.

Secretion of CXC chemokine IP-10 by peripheral blood mononuclear cells from healthy donors and breast cancer patients stimulated with HER-2 peptides.

CXC chemokines play an important role in recruitment of T cells to the site of activation and regulation of angiogenesis. CXC chemokines are secreted by T cells stimulated with cytokines or by established cytotoxic T lymphocyte (CTL) lines at recognition of conventional antigen (Ag), but the activation requirements and the relationship of interferon-gamma (IFN-gamma) inducible protein (IP-10) secretion with IFN-gamma induction in lymphocytes are still unclear. We studied the induction of IP-10 from nonadherent peripheral blood mononuclear cells (PBMC) by IFN-gamma, interleukin-12 (IL-12), and the HER-2 peptide E75, which forms a CTL-defined antigen. We found that IFN-gamma alone was a weak inducer of IP-10 in these cells, whereas IL-12 was a significantly stronger inducer of IP-10. In the presence of IL-12, the tumor peptide E75 (HER-2, 369-377) was a stronger inducer of IP-10 than was IL-12 alone. E75 and its variants mutated at position 5 could also induce IP-10 in the absence of exogenous IL-12 or IFN-gamma. IP-10 induction by E75 required HLA-A2 presentation and B7-CD28 interactions and was partially inhibited by blocking of CD40-CD40L interactions. These results indicate that presentation of tumor peptides to peripheral T cells can induce a fast chemokine response, which in its early phase may be higher than the IFN-gamma response. This shows that the IP-10 response was independent of any early-phase IFN-gamma response in peripheral T cells. This may be important for understanding the regulation of the balance between chemoattractant chemokines (CC) and CXC chemokines by tumor Ag and may have implications for understanding the mechanisms of polarization of T cells and conditioning of antigen-presenting cells (APC) by tumor antigens.

Growth and antigen recognition of tumor-infiltrating lymphocytes from human breast cancer.

In the present study, we isolated tumor-infiltrating lymphocytes (TIL) from 21 primary solid tumors and tumor-associated lymphocytes (TAL) from 9 malignant effusions, respectively, of breast cancer patients. Significant proliferation and expansion of T cells was observed in 23 of 30 distinct samples. TIL were isolated from primary tumors by either enzymatic digestion or mechanical disruption. The TIL cultures were initiated using OKT3 mAb in the presence of moderate concentrations (25-50 U/ml) of IL-2, followed by 100 U/ml of tumor necrosis factor (TNF)-alpha. TAL were not stimulated with OKT3 mAb, but all were successfully expanded in culture in the presence of IL-2 alone or together with TNF-alpha. Seven of nine distinct TAL grew in culture as predominantly CD4+ lines. In contrast, only 14 of 21 (66%) of primary breast TIL expanded in culture and were predominantly of CD8+ phenotype. Autologous tumor lysis was observed in seven of eight cases tested. Only one of the four TIL tested and one of the four TAL tested preferentially lysed autologous tumor. HER-2 peptide E75 (369-377) was recognized by two TIL lines of the five primary TIL tested and three of the four TAL tested. This suggests that E75 may be recognized by primary breast tumors. This may be of interest in developing vaccine strategies for therapeutic management of breast cancer.

Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type. P53 (Ad-p53).

The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected.