Adenovirus-mediated gene transfer of pathogen-associated molecular patterns for cancer immunotherapy.

The delivery of stimulatory signals to dendritic cells (DCs) in the tumor microenvironment could be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of pathogen-associated molecular patterns (PAMPs), microbial molecules which bind Toll-like receptors and deliver activating signals to immune cells, when expressed in tumor cells using adenoviral (Ad) vectors. In vitro, transduction of A549 tumor cells with Ad vectors expressing either flagellin from Listeria monocytogenes or P40 protein from Klebsiella pneumoniae induced the maturation of human monocyte-derived DCs in co-cultures. In mixed lymphocyte reactions (MLRs), Ad-flagellin and Ad-P40 transduction of tumor cells stimulated lymphocyte proliferation and the secretion of IFN-gamma. In vivo, these vectors were used either as stand-alone immunoadjuvants injected intratumorally or as vaccine adjuvants combined with a tumor antigen-expressing vector. When Ad-PAMPs were administered intratumorally to mice bearing subcutaneous syngeneic B16F0-CAR (cocksackie-adenovirus receptor) melanomas, tumor progression was transiently inhibited by Ad-P40. In a therapeutic vaccine setting, the combination of Ad-MUC1 and Ad-PAMP vectors injected subcutaneously delayed the growth of implanted RenCa-MUC1 tumors and improved tumor rejection when compared with vaccination with Ad-MUC1 alone. These results suggest that Ad-PAMPs could be effective immunoadjuvants for cancer immunotherapy.

Proteomic prediction of disease outcome in cancer : clinical framework and current status.

Better than gene sequencing or quantitative amplification, proteomics tools allow the study of tumor phenotype. Indeed, most current prognostic tests in cancer (carcinoembryonary antigen [CEA], prostate-specific antigen [PSA], CA 19-1, CA 125, alpha-fetoprotein [AFP], etc.) are based on the detection and quantification of single proteins in body fluids. However, a common characteristic of these tests is their relatively low predictive value, so that they are usually complemented with other procedures such as biopsy and/or endoscopy. Recently, improved analytical and bioinformatics tools have driven the attention on pattern recognition approaches rather then single-marker tests for prognostic forecasting. It is expected that predicting metastasization on the basis of tumoral protein patterns will soon be a reality. However, currently available technologies either limit the number of proteins that can be analyzed simultaneously or they are expensive, difficult, and time-consuming. Moreover, the tools adapted for expression proteomics might not be the same as those for prognostic studies that require investigation of protein function over time. We believe that clinical proteomics research designed within a precise clinical and pathology framework should be strongly supported, since many prognostic factors are determined not by the tumor itself, but by the patient, the treatment and the environment.

Enhancement of protective efficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered with nanoparticles.

Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic Escherichia coli heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.

Synthetic biomimetic supra molecular Biovector (SMBV) particles for nasal vaccine delivery.

For the optimal delivery of antigens to mucosal tissues, especially as nasal sprays, protein antigen alone is often not sufficient. A clear need for nasal delivery systems has therefore evolved, especially for Influenza A vaccines. Such technologies will be even more essential for new modern vaccines based on recombinant antigens. Here we describe synthetic biomimetic supra molecular Biovector (SMBV) which have proven in preclinical and clinical evaluation to be suitable candidates for the delivery of nasal vaccines. They also demonstrate the potential to work with multiple antigens and furthermore allow combination with adjuvants. These Biovectors can associate with internal or lipid layer membrane proteins and peptides due to their charged polysaccharide core. The mimicry with viruses is also provided through their size of 60-80 nm, which allows sterilization by filtration. This makes them an ideal tool for the development of modern nasal vaccines, as they have shown to be able to induce the desired types of humoral immunity (serosal and mucosal immunity, IgA and IgG antibodies) as well as cellular immunity (CD4 and CD8 responses).

Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine.

To optimize polynucleotide vaccinations for protective antitumor immunity we used a self-replicating RNA vaccine in which Semliki Forest virus replicase drives RNA expression of the lacZ gene coding for beta-galactosidase as model tumor-associated antigen (TAA). This was compared with replicase-deficient control RNA and with lacZ DNA plasmids with respect to gene expression in vitro and in vivo and for vaccination using the mouse ear pinna as an optimal immunization site. In vitro, the highest expression was observed with self-replicating RNA. Gene expression following pinna inoculation of either non-replicating DNA plasmids or self-replicating RNA was similar, lasting for 2-3 weeks. Higher antibody responses were obtained with RNA than with DNA. beta-Gal peptide specific CTL memory responses to lacZ DNA or RNA lasted for more than 6 weeks while respective responses induced by lacZ-transfected tumor cells lasted for only 2 weeks. To achieve a protective response against lacZ tumor cells with self-replicating RNA about a 100-fold lower dose of polynucleotide was sufficient in comparison to DNA. The extent of protective antitumor immunity not only depended on the gene dose used for vaccination, but also on the aggressiveness of the lacZ-transfected tumor line used for challenge. In comparison to lacZ-transfected tumor cells as vaccines, polynucleotide vaccination also demonstrated superiority with regard to cross-protection. Protective antitumor immunity could be strongly increased upon co-inoculation of lacZ DNA with IL-2 DNA or IL-12 RNA. IL-2 DNA, but not IL-12 RNA, also augmented the CTL response while IL-12 RNA, but not IL-2 DNA, reduced the antibody response. These results demonstrate efficient protective antitumor immunity after intra-pinna lacZ TAA polynucleotide vaccination and show additional immunomodulatory effects by co-administration of cytokine polynucleotides.

Immunological and molecular characterization of an aggressive murine lymphoma variant: modulation in vitro and in vivo.

The highly metastatic murine ESb-L lymphoma was analyzed with respect to its possible origin and phenotype modulation. By determining the methylation status of the CD8 gene an early thymic origin of the ESb-L lymphoma cells is suggested. It revealed that the precursors of ESb-L cells had at least one CD8 allele expressed during their development. ESb-L tumor cells were found to express ICAM-1, ICAM-2, VLA-4 and Mel14 as adhesion molecules and homing receptors and CD25, CD69 and CD124 (HSA) as T-cell related activation markers. PCR analysis revealed that ESb-L tumor cells express a Th2-like cytokine pattern with mRNAs for IL-4, IL-5, IL-6, IL-10 and IL-13, but not for IL-2 and IFNgamma. In addition mRNA for TNFalpha, LT, IFNalpha and the chemokines MIP1alpha and MIP1beta was found. The expression of the adhesion molecules ICAM-1, ICAM-2, VLA-4 and of the T-cell activation marker CD25 on ESb-L tumor cells could be upregulated by incubating the cells with 10 ng/ml TNFalpha. For CD25 this effect was confirmed also at the mRNA level. Using the lacZ transduced T-cell lymphoma ESb-L-CI we were able to re-isolate live tumor cells from the primary site or from a metastasized liver and to investigate their phenotype ex vivo. MIP1alpha mRNA expression was strongly reduced in ex vivo isolated tumor cells as compared to in vitro grown cells indicating the modulatory role of the tumor microenvironment. The presented data suggest possible roles of TNFalpha and/or other microenvironmental factors modulating the expression of molecules involved in cell migration and adhesion thereby influencing cancer metastasis.

Immunization with human immunodeficiency virus type 1 rgp120W61D in QS21/MPL adjuvant primes T cell proliferation and C-C chemokine production to multiple epitopes within variable and conserved domains of gp120W61D.

Human immunodeficiency virus type 1 (HIV-1) gp120W61D-specific T cell lines (TCL) were generated from an HIV-1-seronegative volunteer who received rgp120W61D in QS21/MPL adjuvant with emulsion. TCL were challenged with pools of consecutive, overlapping peptides spanning the gp120W61D sequence and then with the individual peptides of the immunostimulatory pool. T cell epitopes were found within both variable and conserved domains, and there was no evidence of a single immunodominant epitope. The two most frequently recognized peptides were located in the C1 domain and in the C-terminal region of the V3 loop. Several TCL were shown to recognize multiple peptides from nonoverlapping regions. Peptides from both conserved and variable domains were capable of inducing MIP-1alpha, MIP-1beta, and RANTES production. When tested against the equivalent peptide from the HIV-1IIIB sequence, however, TCL were able to tolerate only minor conserved changes in the amino acid sequence.

Superiority of the ear pinna over muscle tissue as site for DNA vaccination.

Three different vaccination sites were compared for efficiency of immunization with naked DNA. Using the bacterial lacZ gene as a model, all three sites of the mouse (skeletal muscle, dermis of abdominal skin or of the ear pinna) could express the gene product beta-gal but varied in expression time with muscle tissue showing the longest expression. Expression time, however, did not correlate with immune response intensity. The ear pinna was by far the most effective and muscle the least effective priming site for specific humoral and cytotoxic T cell-mediated immune responses. Following intra-pinna DNA inoculation, beta-gal expressing cells were detectable around the injection site and in the major draining lymph node. Efficiency of immunization was also dependent on the promoter and expression vector used. The cytomegalus virus promoter driven pCMV beta vector was superior to the Moloney murine leukemia virus LTR driven BAG vector. LacZ DNA immunization was also compared with cell-based vaccination with lacZ-transfected tumor cells, in which case again the pinna was the best site for inducing strong immune responses. Tumor-specific T cell responses could also be well induced in the pinna, leading to cytotoxic T lymphocyte induction and protective antitumor immunity. Thus, the pinna was found to be a privileged site for induction of antitumor responses and for genetic immunization, an important finding of immediate practical and potential future clinical implications.

Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance.

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2 x D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2 x D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.

A clinically relevant HIV-1 subunit vaccine protects rhesus macaques from in vivo passaged simian-human immunodeficiency virus infection.

OBJECTIVES: To investigate whether immunization with recombinant HIV-1 envelope protein derived from a clinical isolate could protect macaques from infection with an in vivo passaged chimeric simian-human immunodeficiency virus (SHIV). DESIGN AND METHODS: A total of 16 animals were studied from which three groups of four animals were immunized with vaccine formulations of the CC-chemokine receptor-5-binding recombinant gp120 of HIV-1W6.1D. Four weeks after the last immunization, all 16 animals were intravenously challenged with in vivo passaged SHIV derived from the same HIV-1 group B clinical isolate (W6.1D) as the vaccines. RESULTS: Vaccine protection from infection was demonstrated in 10 out of 12 macaques immunized with recombinant gp120. Complete protection from infection was achieved with all of the animals that received the SBAS2-W6.1D formulation, a potent inducer of both T-cell and humoral immune responses. Partial protection was achieved with SBAS1-W6.1D, a formulation based on immunomodulators known to induce T-cell responses in humans. In vaccinated animals that were infected, virus load was reduced and infection was delayed. CONCLUSIONS: In a relatively large number of primates, vaccine efficacy was demonstrated with a clinically relevant HIV-1 vaccine. These results reveal that it is possible to induce sterilizing immunity sufficient to protect from infection with SHIV which was passaged multiple times in vivo. Our findings have implications for current HIV-1 clinical vaccine trials and ongoing efforts to develop safe prophylactic AIDS vaccines.

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response.

This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2-3) peak of interferon gamma (INF-gamma), interleukin-2 (IL-2), IL-2 receptor alpha (IL-2Ralpha) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-gamma could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Ralpha and IFN-gamma responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/ 2 mice was a delay in the latter in the IL-2, IL-2Ralpha and IFN-gamma responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning.

Dynamic expression changes in vivo of adhesion and costimulatory molecules determine load and pattern of lymphoma liver metastasis.

Although intradermal primary tumor growth and spontaneous liver metastasis of ESbL-lacZ lymphoma in syngeneic DBA/2 mice are progressive and malignant, they are characterized by a transient plateau period with a constant tumor diameter and a low number of metastasized cells in the liver. This period, which was shown to be immune dependent, was followed by a second expansion phase characterized by a preferential localization of tumor cells in the periportal areas of liver lobules (mosaic phenotype). To elucidate possible mechanisms leading to the plateau period as well as for the mosaic-like metastasis pattern, we investigated, using flow cytometry analysis, alterations in costimulatory and adhesion molecule expression in liver sinusoidal cells as well as in tumor cells isolated directly ex vivo throughout the kinetics of metastasis. In tumor and sinusoidal cells, we found up-regulation in the expression of MHC class II and B7 molecules during the plateau period. These molecules, which facilitate cell-mediated immune responses, were again down-regulated during the final exponential tumor growth and metastasis. In the final expansion phase, in which the mosaic phenotype of liver metastasis is seen, we detected a significant increase of leukocyte function-associated antigen-1/intercellular adhesion molecule-1 expression in both tumor and sinusoidal cells, suggesting tumor cell-sinusoidal cell interactions. vascular cell adhesion molecule-1/very late activated antigen-4 did not show any modification during the whole metastatic process. In vivo application of monoclonal antibodies directed to leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 appeared to block the spread of metastasis, while no effect was seen with monoclonal antibodies directed to vascular cell adhesion molecule-1 and very late activated antigen-4. This study reveals in situ expression changes of cell surface molecules in tumor and host cells during metastasis. The changes seen during the plateau phase and during the second expansion phase differ, suggesting associations with mechanisms of immune control and tumor immune evasion, respectively.

Synergistic role of type I interferons in the induction of protective cytotoxic T lymphocytes.

Differentiation of cytolytic T cells can be supported by type I and type II interferons (IFN). To characterize the role of type I interferons further we tested the role of recombinant IFN-alpha and IFN-beta on the induction of a weak immune response, against a low immunogenic tumor, which has been shown to be increased by IFN. Both type I interferons IFN-alpha and IFN-beta were able to support the differentiation of cytolytic T lymphocytes (CTL). In case of IFN-alpha no correlation with the antiviral activity could be seen by comparison of IFN-alpha1 and IFN-alpha4. The maximal in vitro effects were achieved with very low concentrations in the range of 1-100 IU/ml. IFN-alpha showed the strongest effects, if added in the early phase of the mixed leukocyte culture, whereas IFN-beta was most effective when given at the last day the culture. In combination, both IFNs gave additional/synergistic effects, whereby addition of IFN-alpha at day 0 and IFN-beta at day 4 led to maximal specific CTL responses. In vivo augmentation of the anti-tumor immune response by both types of IFNs supported the in vitro findings and also the synergistic effect of both types of IFNs could be demonstrated. Therefore we propose that IFN-alpha is relevant in the induction of CTL responses, i.e., the conversion of precursor T cell into mature cells and growth promotion whereby IFN-beta might trigger the lytic machinery of the cells and promote differentiation. This synergistic efficacy is also operative in tumor rejection.

The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1.

The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4-derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.

Deficiency in T cell responses of human fetal lymph node cells: a lack of accessory cells.

Lymph node and spleen cells of human foetuses from the 18th to the 24th week of gestation were analysed with regard to their phenotypes and their functional capacities. Fetal mesenteric lymph nodes contain high percentages of CD45RA+ T cells and few B cells and monocytes, whereas the fetal spleen is comprised of equal numbers of T and B cells as well as monocytes/macrophages. Functional analysis of lymph node T cells revealed a lack of proliferative response to PHA or CD3 specific mAb, despite induction of expression of the activation marker CD69. Proliferation of LN cells and thymocytes was observed upon addition of exogenous IL-2. An allogeneic EBV transformed tumour cell line, known to be an effective antigen presenting cell, could induce proliferation of LN cells without exogenous IL-2 and fetal spleen cells could proliferate in response to all stimuli tested without additional IL-2. Splenic non-T cells could restore the proliferation of lymph node cells as efficiently as IL-2 or the EBV transformed B cell line. Separated B cells were more effective than plastic adherent cells on a per cell basis. Naivity of the fetal immune system is therefore not only reflected by the expression of markers representative for naive lymphocytes but can also be due to the absence of potential accessory cells in the different lymphoid organs.

Promotion of experimental liver metastasis by tumor necrosis factor.

Models for experimental metastasis were established to investigate the influence of rmTNF on tumor-colony formation in the liver. Highly metastatic lymphoma tumor cells were either injected i.v. or inoculated s.c. to form spontaneous metastases. In both systems, administration of rmTNF to the animals led to significant enhancement of the number of liver metastases in comparison with control groups. The number of metastatic tumor-cell colonies at an early stage of metastasis was increased, as well as the number of surface metastases in a late stage. Consequently, TNF-treated animals revealed a higher mortality. The optimal time for TNF to exert this metastasis-enhancing effect was found to be 7 days after tumor inoculation. In vitro adhesion of the lymphoma tumor cells to a mouse endothelioma cell line was strongly inhibited by monoclonal antibodies interfering with the interaction of VCAM-1 with VLA-4. These results support and extend earlier results with a fibrosarcoma lung colonization model. In addition, they show that stimulation of the immune system in tumor-bearing hosts activates tumor-promoting pathways, in addition to having possible beneficial effects.

Pattern and load of spontaneous liver metastasis dependent on host immune status studied with a lacZ transduced lymphoma.

Detection of disseminated leukemia within organ is often very difficult and might lead to underestimation of the metastatic load. Therefore, we transduced the mouse ESb T lymphoma with the bacterial lacZ gene, which allowed us to follow metastasis at the single cell level. Intradermal primary tumor growth of lacZ transduced ESbL cells (L-CI.5s) comprised three phases: an initial expansion phase (day 0 to 9, increase from 0 to 8 mm, tumor diameter), a plateau phase (day 9 to 20, constant diameter of 8 mm and necrosis), and a second expansion phase (day 20 to 30, increase from 8 to 15 mm). Liver metastasis could already be detected at day 3 and maintained at that level until day 23, where exponential expansion started. A distinct mosaic-like metastasis pattern developed, with preferential localization of tumor cells to the periportal areas of the liver in immunocompetent animals. In contrast, in immunocompromised mice, primary tumor growth and metastasis were progressive and metastasis appeared as diffuse or focal/clustered. Healthy animals surviving a tumor cell inoculum of a variant cell ESbL-CI.5) with a reduced metastatic potential carried low levels of possibly dormant tumor cells in the bone marrow. Thus, this study showed that host immunocompetence determines to a large extent kinetics and load of spontaneous liver metastases and even influences the pattern and localization of disseminated lymphoma cells.

Scattered micrometastases visualized at the single-cell level: detection and re-isolation of lacZ-labeled metastasized lymphoma cells.

To study metastasis at the single-cell level we transduced highly metastatic ESb lymphoma cells with a retroviral expression vector containing the lacZ (bacterial beta-galactosidase) gene. This allowed single ESb-lacZ tumor cells to be detected in infiltrated target organs by means of X-Gal staining. Despite expression of the lacZ gene, the tumor cells were still tumorigenic, highly metastatic, unchanged in phenotype and therefore comparable to parental ESb cells. After spontaneous metastasis, whole-organ staining revealed metastatic foci at the surface of the liver. In histological liver sections, metastatic clusters and single dispersed tumor cells could be detected. In contrast to whole-organ staining, histological examination revealed scattered distribution of tumor cells throughout the organ, which was not evident with parental ESb cells. In addition, clusters with diffuse or dense (focal) appearance were found, in correlation with the whole-organ staining. Expression of the foreign lacZ gene allowed the metastatic spread of tumor cells to liver and spleen to be quantified approximately by FACS analysis. Furthermore, it was shown that the newly expressed beta-gal was expressed not only intercellularly but also at the cell surface. There it could be recognized by MAbs and cytotoxic T-cells (CTL). beta-gal did not affect CTL recognition of the ESb tumor-associated antigen. In conclusion, lacZ could be used as a genetic marker for a highly metastatic lymphoma, to define scattered metastatic spread in the liver at the single-cell level and to quantify the tumor load by FACS analysis.

Viral hemagglutinin augments peptide-specific cytotoxic T cell responses.

In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50-63 (NP 50-63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk-) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDNA of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk- cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.

Tumour-specific CTL response requiring interactions of four different cell types and recognition of MHC class I and class II restricted tumour antigens.

This study demonstrates that a syngeneic specific cytotoxic T lymphocyte (CTL) response to a class I major histocompatibility complex (MHC) positive tumour requires dual processing and recognition of tumour antigens. One type of antigen is processed and expressed in association with class I MHC at the surface of intact tumour cells. It is recognized by CD8 alpha, beta TCR CTL in vitro and by protective immune T cells in vivo and thus functions as a tumour-associated transplantation antigen (TATA). The other type of antigen is processed and expressed by distinct host APC in association with class II MHC. This is recognized by immune CD4 T cells which function as essential helper cells in the generation of the CD8 CTL response. These conclusions are supported by cell depletion and reconstitution experiments as well as by blocking experiments with monoclonal antibodies using the highly metastatic class II negative murine lymphoma ESb as a model system. The existence of two types of cognate T cell responses in a syngeneic anti-tumour response was directly proved by the establishment of two types of tumour specific T cell lines which required as co-stimulator either MHC class II positive APC or IL-2. In suboptimal mixed lymphocyte tumour cell cultures either of these co-stimulator functions was found to be limiting the overall anti-tumour CTL response. The generation of the tumour specific CTL response could be blocked by monoclonal antibodies against all the molecules involved in the cognate interactions (i.e. class I MHC, CD8, class II MHC, CD4 and TCR) but not by anti-CD2 or anti-IgG. The strict requirement for helper cells and APC could be bypassed by the addition of recombinant IL-2 but optimal triggering of CD8 CTL-precursor required viable tumour stimulator cells. This well characterized in vitro assay may be useful (i) for monitoring the immune status of CD4 and CD8 immune T cells separately, for instance of tumour bearing and/or treated animals and (ii) for the development and testing of potent tumour cell vaccines with T cell stimulatory and/or co-stimulatory activities.