Michael Heike

T cell specificity and cross reactivity towards enterobacteria, Bacteroides, Bifidobacterium, and antigens from resident intestinal flora in humans

BACKGROUND T cell responses to normal intestinal bacteria or their products may be important in the immunopathogenesis of chronic enterocolitis. AIMS To investigate the T cell specificity and cross reactivity towards intestinal bacteria. PATIENTS/METHODS T cell clones were isolated with phytohaemagglutinin from peripheral blood and biopsy specimens of inflamed and non-inflamed colon from five patients with inflammatory bowel disease (IBD) and two controls. T cell clones were restimulated with anaerobicBacteroides andBifidobacteria species, enterobacteria, and direct isolates of aerobic intestinal flora. T cell phenotype was analysed by single-cell immunocyte assay. RESULTS Analysis of 96 T cell clones isolated from peripheral blood and biopsy specimens from two patients with IBD showed that bothBifidobacterium andBacteroides species specifically stimulate proliferation of CD4+TCRαβ+ T cell clones from both sites and that cross reactivity exists between these anaerobic bacteria and different enterobacteria. Analysis of 210 T cell clones isolated from three patients with IBD and two controls showed that indigenous aerobic flora specifically stimulate T cell clones from peripheral blood and biopsy specimens from a foreign subject. Some of these flora specific T cell clones were cross reactive with defined enterobacteria. In addition, T cell clones stimulated by their own indigenous aerobic flora were identified in patients with IBD. CONCLUSION Immune responses to antigens from the intestinal microflora involve a complex network of T cell specificities.

Heat shock protein vaccines against cancer

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Tumor-specific cell surface expression of the -KDEL containing endoplasmic reticular heat shock protein gp96

Heat shock protein (HSP) gp96/grp94 contains a signal peptide at the amino terminus and a ‐KDEL sequence at the carboxy terminus and is a major component of the lumen of the mammalian endoplasmic reticulum (ER). We show, by a number of immunolocalization methods using light and electron microscopy, that a significant proportion of intact gp96 molecules is also expressed on the cell surface. Surface gp96 molecules truly represent surface expression and do not result from adventitious deposition of gp96 released by dead cells on to the live cells in culture. Cell surface expression of gp96 is enhanced by heat shock and exposure to reducing agents. Gp96 molecules are not released from plasma membranes by repeated salt washes, and gp96 is not an integral membrane protein. Our observations suggest that gp96 and perhaps other HSPs are anchored to the cell surface as part of larger molecular complexes, which also transport them to the cell surface.

Different Efficiency of Heat Shock Proteins (HSP) to Activate Human Monocytes and Dendritic Cells: Superiority of HSP60

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.

Weekly high-dose leucovorin versus low-dose leucovorin combined with fluorouracil in advanced colorectal cancer: results of a randomized multicenter trial. Study Group for Palliative Treatment of Metastatic Colorectal Cancer Study Protocol 1.

PURPOSE To determine the most effective dose of leucovorin (folinic acid [FA]) within a weekly bolus fluorouracil (FU) schedule, we conducted a randomized multicenter trial to compare therapeutic effects and toxicity of high-dose FA versus low-dose FA combined with FU at equal doses in both treatment groups. PATIENTS AND METHODS Patients with measurable inoperable or metastatic colorectal cancer were randomized to receive weekly FU 500 mg/m2 by intravenous (IV) bolus combined with high-dose FA 500 mg/m2 (group A) or low-dose FA 20 mg/m2 (group B) by 2-hour infusion. RESULTS Of 291 assessable patients (group A, n = 148; group B, n = 143), we observed, in group A, complete response (CR)/partial response (PR) in 32 (21.6%), no change (NC) in 64 (43.2%), and progressive disease (PD) in 52 (35.1%); and in group B, CR/PR in 25 (17.5%), NC in 63 (44.1%), and PD in 55 (38.5%). The median response duration was 24.8 weeks in group A and 23.1 weeks in group B. Median progression-free intervals were 29.3 weeks (group A) and 30 weeks (group B). The median survival time was 55.1 weeks in group A and 54.1 weeks in group B. Overall toxicity was moderate. Mild nausea and vomiting, and stomatitis were common side effects in both groups. The incidence of World Health Organization (WHO) grade III/IV diarrhea was significantly higher in group A (40 v 23 patients). Severe side effects were observed only in a minority of patients in both arms. WHO grade IV diarrhea was observed in seven patients: four in group A and three in group B. The rate of toxicity-related adjustments of dose and schedule was comparable in both groups. CONCLUSION High-dose FA/FU is not superior to low-dose FA/FU within a weekly treatment schedule. Response rates and survival were comparable in both treatment arms. Treatment-related toxicity was higher in group A (high-dose FA). Therefore, low-dose FA combined with weekly FU may be considered the preferred treatment for metastatic colorectal cancer.

Heat shock protein-peptide complexes for use in vaccines

The heat shock proteins gp96, HSP70, and HSP90 are complexed to a diverse array of cellular proteins and peptides as a consequence of their chaperone functions. There is good experimental evidence that vaccination with these heat shock protein‐peptide complexes elicit immune responses against chaperoned peptide antigens. As shown with gp96, this requires internalization of the heat shock protein‐peptide complexes by macrophages and processing of the chaperoned peptides for class I restricted presentation. Via this process, primarily CD8+ antigen‐specific T cells are primed by gp96 vaccination. This might represent a general mechanism for priming of MHC‐class I restricted T cells by professional antigen‐presenting cells. At least gp96 has been shown to be associated with peptides that are not selected by the MHC haplotype of the harboring cell. This allows for immunization with gp96‐peptide complexes across MHC barriers, for example against shared tumor antigens or viral antigens. J. Leukoc. Biol. 60: 153–158; 1996.

Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells

A limiting-dilution assay was developed and used to determine the frequency of autologous tumor-reactive cytotoxic T lymphocytes (CTL) in peripheral blood of a melanoma patient MZ2, who has been free of detectable disease since several years. In this patient, the frequencies of tumor-reactive CTL spontaneously varied only by a factor of 1.5. After vaccinations with autologous mutagenized and lethally irradiated tumor cells a two- to tenfold increase in frequencies of tumor-reactive CTL was found within the first 2 weeks. Thereafter, CTL frequencies returned to values measured prior to vaccinations. We conclude, that the limiting-dilution assay applied in this study can detect changes in the T cell response to autologous tumor cells. The frequency of tumor-reactive CTL determined with this approach can serve as an immunological parameter for monitoring the T cell response to autologous tumor cells in individual cancer patients receiving tumor cell vaccinations.

Stress protein/peptide complexes derived from autologous tumor tissue as tumor vaccines.

Vaccination of inbred mice with tumor-derived stress proteins hsp70, hsp90, and gp96/grp94 elicits a protective immunity to the tumor from which the vaccine was purified. There is now comprehensive experimental evidence that the antigenicity of tumor-derived hsp70, hsp90, and gp96 preparations results from diverse arrays of endogenous peptide antigens complexed with these stress proteins. Vaccination with tumor-derived stress protein/peptide complexes leads to their uptake and processing by professional antigen-presenting cells and to presentation of associated tumor peptide antigens to cytotoxic T cells. This induces a tumor-specific cytotoxic T cell response. The attractiveness of the concept of using tumor-derived stress proteins as vaccines is derived from two observations: (i) tumor stress protein vaccines mirror the individual antigenicity of a tumor, which results from random mutations due to genetic instability; and (ii) stress proteins represent powerful adjuvants for the peptide antigens complexed to them.

Oncolytic parvovirus H1 induces release of heat-shock protein HSP72 in susceptible human tumor cells but may not affect primary immune cells.

Certain autonomous parvoviruses preferentially replicate in and kill in vitro-transformed cells and may reduce the incidence of spontaneous and implanted tumors in animals. Hence, these viruses and their derivatives are currently under evaluation as antitumor vectors. However, the mechanisms underlying their tumor-suppressing properties are not yet understood. We asked whether the lytic parvovirus H1 may enhance the immunogenicity of infected tumor cells. Out of human melanoma and gastrointestinal tumor cells, we selected the cell line SK29-Mel-1 being very susceptible to H1-induced apoptotic killing. Here, no upregulation of HLA class I and costimulatory molecules could be observed following H1 infection. However, a strong release of the immunogenic signal—the inducible heat-shock protein HSP72, but not constitutive HSP73—was observed after H1 infection. The HSP72 release was higher and of longer duration than a conventional heat-shock treatment. We also explored H1 replication and cytotoxicity in human immune cells, as such cells may constitute targets for H1 virus replication. Long-term cultured lymphocytes, monocytes, immature and mature dendritic cells were not susceptible to H1 virus. Altogether, parvovirus-mediated cell killing may in vivo enhance tumor immunogenicity by HSP72 release and thus contribute to the antitumor effect of parvoviruses.

Treatment of advanced pancreatic cancer with 5-fluorouracil, folinic acid and interferon alpha-2A: results of a phase II trial.

Interferon alpha-2a (IFN-alpha) and folinic acid (FA) have been shown to modulate the cytotoxic effects of 5-fluorouracil (5-FU) in the treatment of cancer. A phase II study was initiated to evaluate the effect of a combination of 5-FU/FA/IFN-alpha in patients with advanced pancreatic cancer. Sixty previously untreated patients with advanced adenocarcinoma of the pancreas were treated with 500 mg m-2 FU via an intravenous bolus 1 h after the initiation of a 2 h infusion of 500 mg m-2 FA. Before starting the FA infusion, 6 million units (MU) of IFN-alpha was administered subcutaneously. The treatment was repeated once a week. Of 57 evaluable patients, eight (14%) had a partial response (PR), eight (14%) a minor response (MR) and 28 (49%) no change of disease (NC). Thirteen patients (23%) had progressive disease (PD). The median survival time was 10 months for all patients, 22 months for patients with partial remission and 5 months for patients with progressive disease. Many patients with tumour-related pain whose tumours were affected in terms of PR, MR, NC were free of pain during treatment with this regimen (22/36 patients). The common toxicities observed were fever (56%), nausea (37%) and diarrhoea (33%). These data suggest that biochemical modulation of 5-FU with FA and IFN-alpha has some positive effects in the treatment of pancreatic cancer of moderate toxicity.