Interstitial laser thermotherapy of a rat liver adenocarcinoma.

The aim of this study was to compare interstitial laser thermotherapy with excision of a liver tumour. A dimethylhydrazine-induced adenocarcinoma was implanted into the left lateral lobe of the rat liver, and treatment was performed 8 days later. Rats were treated with resection of the tumour-bearing lobe or underwent interstitial laser thermotherapy, which was performed at a steady-state temperature of 46°C for 30 min, 3 mm from the tumour margin. The incidence and extent of intraperitoneal spread was smaller after laser thermotherapy than after resection, with no difference in local control. Using inoculation of tumour cell suspensions into the lateral and the median lobes of the liver simultaneously and treating the lateral lobe tumour only, we found that laser thermotherapy reduced take and growth of the untreated tumour in the median lobe indicating that laser thermotherapy may induce immunologic effects. It is concluded that interstitial laser thermotherapy reduces spread of liver tumour as compared to resection. It is suggested that this can be at least partly explained by a laser-induced immunologic effect.

Computerized image analysis as a tool to quantify infiltrating leukocytes: a comparison between high- and low-magnification images.

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a x100 or a x12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-gamma (IFN-gamma) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software.(J Histochem Cytochem 49:1073-1079, 2001)

Inhibition of the transcription factors AP-1 and NF-kappaB in CD4 T cells by peroxisome proliferator-activated receptor gamma ligands.

The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, is essential for adipocyte differentiation and glucose homeostasis. PPARgamma has been found recently to regulate macrophage activation in response to mitogens and inflammation. Our study shows PPARgamma to be preferentially expressed in the nuclei of resting T cells and to increase upon activation of T cells by either anti-CD3 and anti-CD28 or phorbol myristyl acetate (PMA). We also found the PPARgamma ligand ciglitizone to attenuate the activation of T cells by inhibiting cytokine gene expression and anti-CD3 and anti-CD28 or PMA-induced proliferative responses. Inhibition of both the proliferative response and inflammatory cytokine expression in CD4 T cells was correlated with suppression of the activated transcription factors AP1 and NF-kappaB. PPARgamma ligands also strongly inhibited SEA-induced Vbeta3 T cell activation in vivo. These results, together with previous findings of the inhibitory effect of PPARgamma ligands on activated macrophages, provide clear evidence for PPARgamma as a negative regulator of the inflammatory activation of both macrophage and T cells. PPARgamma may thus be a potential therapeutic target for the treatment of autoimmunity.

Assembly of tapasin-associated MHC class I in the absence of the transporter associated with antigen processing (TAP).

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and beta(2)-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP.

Nitric oxide synthase inhibitor and IL-18 enhance the anti-tumor immune response of rats carrying an intrahepatic colon carcinoma.

The role of nitric oxide (NO) produced by adherent spleen cells in the systemic immunosuppression developing in tumor-bearing hosts was investigated. After therapeutic immunization of rats carrying an intrahepatic colon carcinoma, H1D2, the spleen cell antitumor immune responsiveness was analyzed. Compared to parallel immunized tumor-free rats, tumor-bearing rats (TB rats) had a greatly reduced proliferative T-cell response to wild-type tumor stimulator cells. The TB rats had a depressed proliferative response to anti-CD3 and to the superantigen SEA. TB rats with small tumors had a stronger response to IL-18-producing H1D2 stimulator cells than to wild type H1D2 cells. This was not the case with TB rats carrying larger tumors. Also the IFN-gamma production and cytotoxicity against the wild-type tumor cells and the NK sensitive YAC cells were depressed in spleen cells of TB rats after 5-day restimulation with wild-type tumor cells. A part of this immunosuppression was mediated by adherent spleen cells, mostly consisting of macrophages. An important mode of action appears to involve their production of an enhanced level of nitric oxide, since the competitive nitric oxide synthase (NOS) inhibitor L-NAME could partially counteract the suppression in vitro. We conclude that NOS inhibitors in combination with immunostimulatory cytokines, such as IL-18, could be useful tools to enhance anti-tumor immune responses in TB rats and therefore to increase the efficiency of immunotherapies.

Vaccination with B16 melanoma cells expressing a secreted form of interleukin-1beta induces tumor growth inhibition and an enhanced immunity against the wild-type B16 tumor.

We have demonstrated previously that gene transfer of the mature human interleukin-1beta (IL-1beta) gene, fused to a signal sequence (ss), into mouse B16 melanoma cells results in an inhibition of their growth in vivo compared with control B16 cells. We here extend these results to show that intraperitoneal vaccinations with irradiated IL-1beta-secreting cells result in protection against subsequent subcutaneous challenge with wild-type (wt) B16 tumor cells in syngeneic C57BL/6 mice. This protection appears to be long-lasting, because rechallenge of cured mice 4 months after the first challenge also demonstrated resistance. In addition, we demonstrate that mice with established wt tumors subjected to therapeutic vaccinations with irradiated B16/ssIL-1beta cells starting 3 days after challenge isografting have a significantly inhibited tumor growth and 25-40% survival at the challenge doses given. In vitro coculture of spleen cells from B16/ssIL-1beta vaccinated animals and wt B16 cells induced an enhanced proliferative response, which correlated with elevated production of IL-2 and interferon-gamma. A significantly enhanced cytolytic activity against B16 wt target tumor cells was observed when spleen cells from B16/ssIL-1beta vaccinated mice were used as effector cells compared with spleen cells from control vaccinated mice. In vitro depletion experiments using anti-asialo GM1 revealed a prominent role for natural killer cells as effector cells. The data suggest that local IL-1beta secretion during the vaccination phase can provoke or augment protective immune responses to B16 melanoma cells, which are otherwise not recorded in mice bearing B16 tumors.

Immunohistochemical analysis of glioma-infiltrating leucocytes after peripheral therapeutic immunization with interferon-gamma-transfected glioma cells.

We have shown previously that rejection of preinduced rat brain tumours is possible following therapeutic immunizations with interferon-gamma (IFN gamma)-transfected glioma cells (N32-IFN gamma). In the present study we have used the same model to evaluate whether quantitative differences in tumour-infiltrating lymphocytes can be detected between animals receiving therapeutic immunizations with either IFN gamma-transfected glioma cells, wild-type glioma cells or no treatment. Since leucocyte transpedesis into the tumour can be anticipated to depend on the state of vascularization, we have mapped the development of microvessels in the tumour in parallel with the leucocyte infiltration. Our results show that microvessels start to form at day 7 and then gradually increase in number and size, indicating the establishment of an extensive vascularization by day 24. Leucocyte infiltration displays a biphasic pattern after tumour grafting. We have therefore studied the infiltration kinetics after an early immunization (1 day after intracerebral isografting) and compared the effects with those of a late immunization (10 days after intracerebral isografting) with N32-IFN gamma or wild-type N32. Our results show (1) an early infiltration of granulocytes 3 days after isografting; (2) a T-cell-receptor-positive (TCR+) T-cell infiltration starting on day 10; (3) a macrophage infiltration starting on day 13; (4) a CD8+ cell infiltration starting on day 13. The proportions of TCR+ T cells, CD8+ cells and natural killer cells differs significantly between animals immunized with N32-IFN gamma and those receiving wild-type N32, when analysed 14 days after immunization at day 10. This difference can only be detected when animals are immunized at later stages of tumour growth. We propose that this could depend on an early-immunization-independent leucocyte infiltration during tumour establishment. This has to be considered when evaluating studies of leucocyte infiltration in experimental tumours.

Association of decreased phosphorylation of ERK-2 with costimulation of rat T cell activation by MEK-1 inhibitors and TGF-beta1.

Transforming growth factor-beta (TGF-beta) is usually known as an immunosuppressive cytokine, but we and others have shown stimulatory effects of TGF-beta on activation of Th2 T-lymphocytes. In the present investigation we have studied the effect of TGF-beta1 on phosphorylation of ERK, a MAP-kinase downstream of the Ras pathway. ERK is phosphorylated by MEK-1 and PD098059 and U0126 are specific inhibitors for this kinase. We demonstrate in the present study that these inhibitors abrogate the inhibitory effect of adh-splc (adherent-spleen cells) on activation of primary rat T-cells and induce a strong costimulatory effect almost as strong as we have previously shown with TGF-beta1. When TGF-beta1 is acting stimulatory on T-cell activation, it decreases phosphorylation of ERK-2 and thereby its activation. To investigate whether TGF-beta1 and MEK-1 inhibitors influence the same pathways, we compared their effects on cytokine profiles associated with SEA-induced rat T cell activation. TGF-beta1 induced IL-10 production, slightly decreased TNF-alpha production and decreased IFN-gamma production. The PD098059 inhibitor decreased both IFN-gamma and TNF-alpha production and together with TGF-beta1, it totally blocked IFN-gamma, TNF-alpha and IL-10 production. Thus TGF-beta1 and PD098059 showed overlapping but not identical effects on the cytokine pattern.

Expression of TGF-beta isoforms, TGF-beta receptors, and SMAD molecules at different stages of human glioma.

Human gliomas express TGF-beta but, so far the expression of downstream mediators has been investigated in only a few cell lines. We have examined tissue specimens of 23 gliomas: 3 astrocytomas grade II (AST), 8 anaplastic astrocytomas grade III (AAST), and 12 glioblastoma multiforme grade IV (GBM). We analyzed the mRNA expression of TGF-beta1, TGF-beta2, TGF-beta3, the TGF-beta receptors type I (TbetaR-I) and type II (TbetaR-II), Smad2, Smad3, and Smad4. mRNA expression of IL-10 and CD95 (FAS/APO-1) were also studied. We detected increased mRNA levels of the 3 TGF-beta isoforms, correlating with the degree of malignancy. TGF-beta3 mRNA was increased, particularly in AST and AAST, while TGF-beta1 and TGF-beta2 mRNAs were strongly expressed in GBM. TGF-beta normally up-regulates the TGF-beta receptors, and TbetaR-I and TbetaR-II showed stronger expression in all gliomas when compared to normal tissues. However, the mRNA expression of Smad2, Smad3, and Smad4 was decreased in GBM. IL-10 mRNA expression was detected in glioma tissues but not in glioma cell lines. No marked increase in the expression of soluble CD95 splicing variants was found in the gliomas compared with normal tissue. However, total CD95 mRNA was elevated among GBM tissues.

Nitric-oxide-dependent systemic immunosuppression in animals with progressively growing malignant gliomas.

The role of nitric oxide (NO) and adherent spleen cells in systemic immunosuppression developing in animals carrying malignant glioma isografts was analyzed. Rats harboring a subcutaneous glioma isograft for 3 weeks were immunized with glioma cells genetically engineered to express IFN-gamma. One week later spleen cells were tested for immune responsiveness in vitro. A decreased cytotoxic activity of NK-cells and T-cells compared to tumor-free animals immunized in parallel was shown. Spleen cell proliferative responses to tumor cells, SEA, and anti-CD3 were all significantly suppressed, as was the production of IFN-gamma and IL-10. Plastic adherent spleen cells from tumor-bearing rats suppressed the SEA-induced proliferative response and the production of IFN-gamma and IL-10 by nonadherent spleen cells from tumor-free rats. A major part of this suppression appears to be dependent on the production of NO because suppression was efficiently counteracted in vitro by the NO-synthase inhibitor N-nitro-l-arginine methyl ester. Moreover, a significantly increased level of nitrite in culture supernatants correlated with the observed suppression. We conclude that the systemic immunosuppression associated with growing gliomas is in part mediated by mechanisms dependent on NO overproduction in adherent spleen cells.

Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptor-mediated proliferation and prolongs survival of activated T cells.

Members of the suppressor of cytokine signaling (SOCS) family were discovered as negative regulators of cytokine signaling by inhibition of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway. Among them, cytokine-induced Src homology 2 (SH2) protein (CIS) was found to inhibit the interleukin 3- and erythropietin-mediated STAT5 signaling pathway. However, involvement of SOCS proteins in other signaling pathways is still unknown. This study shows that the expression of CIS is selectively induced in T cells after T cell receptor (TCR) stimulation. In transgenic mice, with selective expression of CIS in CD4 T cells, elevated CIS strongly promotes TCR-mediated proliferation and cytokine production in vitro, and superantigen-induced T cell activation in vivo. Forced expression of CIS also prolongs survival of CD4 T cells after TCR activation. Molecular events immediately downstream from the TCR are not changed in CIS-expressing CD4 T cells, but activation of mitogen-activated protein (MAP) kinase pathways by TCR stimulation is significantly enhanced. Together with the increased MAP kinase activation, a direct interaction of CIS and protein kinase Ctheta was also demonstrated. These results suggest that CIS is one of the important regulators of TCR-mediated T cell activation. The functions of CIS, enhancing TCR signaling and inhibiting cytokine signaling, may be important in the regulation of immune response and homeostasis.

Tapasin is required for efficient peptide binding to transporter associated with antigen processing.

The transporter associated with antigen processing (TAP) binds peptides in its cytosolic part and subsequently translocates the peptides into the lumen of the endoplasmic reticulum (ER), where assembly of major histocompatibility complex (MHC) class I and peptide takes place. Tapasin is a subunit of the TAP complex and binds both to TAP1 and MHC class I. In the absence of tapasin, the assembly of MHC class I in the ER is impaired, and the surface expression is reduced. To clarify the function of tapasin in the processing of antigenic peptides, we studied the interaction of peptide and TAP, peptide transport across the membrane of the ER, and association of peptides with MHC class I molecules in the microsomes derived from tapasin mutant cell line 721.220, its sister cell line 721.221 expressing tapasin, and their HLA-A2 transfectants. The binding of peptides to TAP in tapasin mutant 721.220 cells was significantly diminished in comparison with 721.221 cells. Impaired peptide-TAP interaction resulted in a defective peptide transport in tapasin mutant 721.220 cells. Interestingly, despite the diminished peptide binding to TAP, the transport rate of TAP-associated peptides was not significantly altered in 721.220 cells. After transfection of tapasin cDNA into 721.220 cells, efficient peptide-TAP interaction was restored. Thus, we conclude that tapasin is required for efficient peptide-TAP interaction.

Effects of transforming growth factor beta1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor alpha.

The cytokine transforming growth factor beta-1 (TGFbeta1), was transfected into a TGFbeta1-negative rat colon carcinoma. The growth of isografts of TGFbeta1-expressing tumors was compared to that of vector control transfectants. The TGFbeta1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFbeta1-transfected tumor tissue had significantly greater infiltration of both CD4+ and CD8+ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFbeta1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor alpha (TNFalpha) than did TIL from the vector control tumor. The TGFbeta1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFbeta1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFalpha on tumor proliferation in vitro. These results suggest that TGFbeta1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth.

Comparison of 125I- and (111)In-labeled monoclonal antibody BR96 for tumor targeting in combination with extracorporeal immunoadsorption.

Extracorporeal whole blood immunoadsorption (ECIA) accelerates the clearance of radiolabeled monoclonal antibodies (mAbs) without significantly affecting tumor uptake by removing the excess of these mAbs from the blood, thus increasing tumor:normal tissue (T:N) ratios. The present study is focused on comparing the capacity of ECIA in tumor targeting with the same mAb (chiBR96-biotin) labeled with either (111)In or 125I. Forty-five Brown Norwegian rats with syngeneic rat colon carcinoma isografted both in liver and intramuscularly were used. chiBR96 is a highly tumor-specific mAb directed against the Lewis-type antigen. ECIA of whole blood was started 15 h after the injection of 125I- or (111)In-labeled BR96-biotin. The procedure lasted for 2 h and was repeated for (111)In-labeled BR96-biotin in a few rats after 3 or 24 h. ECIA reduced the whole body activity by the same magnitude (between 39% and 52%), irrespective of whether (111)In- or 125I-labeled chiBR96 was used. A similar observation was also made for the reduction in blood radioactivity after ECIA (79-94%). Time-activity curves during ECIA showed that the major reduction (approximately 85%) in blood radioactivity occurred during the first 45-60 min. Repeating the ECIA with (111)In-BR96 caused only an additional minimal reduction of blood activity, whereas a further reduction of whole body activity of 14-20% was achieved. The T:N uptake ratios were significantly enhanced immediately after ECIA with (111)In- or 125I-labeled chiBR96. Due to greater accumulation of (111)In-BR96 in tumors, a long-term improvement in T:N ratios was obtained after ECIA compared with 125I-labeled BR96. Our results therefore indicate that (111)In/(90Y)-labeled BR96-biotin could be more advantageous than 125I/131I for radioimmunotargeting/radioimmunotherapy in combination with ECIA due to better activity retention by the tumor.

HERV-F (XA34) is a full-length human endogenous retrovirus expressed in placental and fetal tissues.

The complete sequence of XA34 was identified from a 107 kb genomic clone originating from the human chromosome 7q31.1-q31.3. The 7.1 kb human endogenous retrovirus (HERV) contains LTR's, gag, pol and env, and a pol sequence which is identical to the 2.3 kb XA34 cDNA clone which we previously isolated from a human glioma cDNA library (Widegren et al., 1996). The HERV is located in a reversed orientation within an intron-sequence of a gene similar to mouse adseverin(D5). The gag and protease regions are intact. However, the pol and env regions are truncated by a deletion which removes the C-terminal end of the integrase and the complete surface protein. The HERV sequence is bordered by a five base-pair direct repeat and has the TG...CA structure. Over the complete HERV genome, XA34 is very similar to members of the HERV-F family and shares the same primer binding site which is homologous to phenylalanine (F) tRNA. Therefore, XA34 is termed HERV-F(XA34). HERV-F(XA34) has an open reading frame (ORF) of 1000 bp in the gag region which starts with Met-Gly in a favorable context and stops in the capsid protein. A strong mRNA expression of HERV-F(XA34) is demonstrated in placental tissue, mainly residing in two transcripts of approximately 7.5 and 8.5-9 kb respectively. Analyses of expressed sequence tags (ESTs) have identified the expression of HERV-F(XA34) sequences in placental tissue, fetal liver/spleen, olfactory epithelium and in an epithelial skin tumor. EST analysis has also identified splice variants of HERV-F(XA34), in which the gag, pol and most of the env regions are spliced out. These splice variants contain a short ORF encoded in the region from the C-terminal portion of env to the 3'-LTR. In addition, ESTs identical to HERV-Fb have been identified in retinal, fetal liver/spleen and brain tissue as well as Jurkat cells. The analyses indicate that the 5'-LTR of HERV-Fb may function as an alternative poly A site of a Krüppel related zinc finger gene (ZNF195).

Transforming growth factor-beta1, a strong costimulator of rat T-cell activation promoting a shift towards a Th2-like cytokine profile.

TGF-beta is a known regulator of hematopoietic cells. In this study we suggest a major role of adherent spleen cells (adh-splc), to convert an inhibitory effect of TGF-beta1 on T-cell activation into a stimulatory effect. We show that interaction of TGF-beta1 with adh-splc induces a costimulatory effect on T-cell proliferation. This costimulatory signal requires the adh-splc to be in physical contact with the T-cells. Presence of adh-splc results in a shift towards a Th2-like response with a cytokine profile of increased IL-10 and decreased IFN-gamma. In the adh-splc population the increase of IL-10 is most pronounced at start of activation, whereas in the T-lymphocyte population, IL-10 increases at the end of culture. The suppression of the IFN-gamma production by TGF-beta1 is shown to be an important mechanism by which TGF-beta enhances proliferation of Th2 lymphocytes.

Peptide-bound major histocompatibility complex class I molecules associate with tapasin before dissociation from transporter associated with antigen processing.

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8 T cells. The peptides are generated in the cytosol, then translocated across the membrane of the endoplasmic reticulum by the transporter associated with antigen processing (TAP). TAP is a trimeric complex consisting of TAP1, TAP2, and tapasin (TAP-A) as indicated for human cells by reciprocal coprecipitation with anti-TAP1/2 and anti-tapasin antibodies, respectively. TAP1 and TAP2 are required for the peptide transport. Tapasin is involved in the association of class I with TAP and in the assembly of class I with peptide. The mechanisms of tapasin function are still unknown. Moreover, there has been no evidence for a murine tapasin analogue, which has led to the suggestion that murine MHC class I binds directly to TAP1/2. In this study, we have cloned the mouse analogue of tapasin. The predicted amino acid sequence showed 78% identity to human tapasin with identical consensus sequences of signal peptide, N-linked glycosylation site, transmembrane domain and double lysine motif. However, there was less homology (47%) found at the predicted cytosolic domain, and in addition, mouse tapasin is 14 amino acids longer than the human analogue at the C terminus. This part of the molecule may determine the species specificity for interaction with MHC class I or TAP1/2. Like human tapasin, mouse tapasin binds both to TAP1/2 and MHC class I. In TAP2-mutated RMA-S cells, both TAP1 and MHC class I were coprecipitated by anti-tapasin antiserum indicative of association of tapasin with TAP1 but not TAP2. With crosslinker-modified peptides and purified microsomes, anti-tapasin coprecipitated both peptide-bound MHC class I and TAP1/2. In contrast, anti-calreticulin only coprecipitated peptide-free MHC class I molecules. This difference in association with peptide-loaded class I suggests that tapasin functions later than calreticulin during MHC class I assembly, and controls peptide loading onto MHC class I molecules in the endoplasmic reticulum.

Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-gamma, interleukin-7, or B7-1-transfected tumor cells.

Progress in the definition of the roles of various costimulators and cytokines in determining the type and height of immune responses has made it important to explore genetically altered tumor cells expressing such molecules for therapeutic immunizations. We have studied the effect of therapeutic subcutaneous (s.c.) immunizations on the growth of preexisting intracerebral brain tumor isografts in the rat. Transfectant glioma cell clones expressing either rat interferon-gamma (IFN-gamma), rat interleukin-7 (IL-7), or rat B7-1 were selected. After irradiation (80 Gy) the clones were used for immunization (administered in up to four s.c. doses in a hind leg over 14-day intervals starting 1 day after the intracranial isografting of the parental tumor). Significant growth inhibition of the intracerebral parental tumors was induced by transfectants expressing IFN-gamma and IL-7, respectively. The strongest effect was observed with IFN-gamma-expressing cells, resulting in cures in 37% of the males and in 100% of the females. Immunization with IL-7 had a similar, strong initial effect, with significantly prolonged survival in the majority of the rats but a lower final cure rate (survival for >150 days). The B7-1-expressing tumor clones induced cures in seven of eight female rats; however, no cures were seen in the male rats. It was also shown that the B7-1-expressing cells were themselves strongly immunogenic in female rats, requiring high cell numbers to result in a progressively growing tumor upon s.c. isografting; this was not the case in male rats. As a whole, the results imply that despite the unfavorable location of intracerebral tumors, therapeutic s.c. immunizations with certain types of genetically altered tumor cells can induce complete regressions with permanent survival and without gross neurological or other apparent signs of brain damage. The present results demonstrate complete regressions when immunizations are initiated shortly after intracranial isografting, when the intracerebral tumor is small.

The three isoforms of transforming growth factor-beta co-stimulate rat T cells and inhibit lymphocyte apoptosis.

In this study the three different mammalian isoforms of transforming growth factor-beta (TGF-beta) were compared with regard to their effect on the response of rat T lymphocytes to the superantigen, staphylococcal enterotoxin A (SEA). All different isozymes were found to increase the proliferative response of rat T lymphocytes, which was accompanied by a significantly lower percentage of apoptotic cells than proliferation in the absence of TGF-beta. The same effect of TGF-beta was observed on the generation of apoptotic cells in an allo response (mixed lymphocyte reaction). TGF-beta2 and TGF-beta3 were three to 10-fold more potent than TGF-beta1 as co-stimulators of T lymphocytes, and equal in decreasing the percentage of apoptotic T cells. TGF-beta1 reduced the frequency and the number of cells undergoing apoptosis in T cells and, to an even higher degree, among B lymphocytes. TGF-beta did not seem to affect the production of the apoptosis inducer, tumour necrosis factor-alpha (TNF-alpha), neither at the mRNA level nor at the protein level. Neutralizing antibodies against the cytokine, TNF-alpha, decreased the percentage of apoptotic cells among T cells responding to SEA, both in the absence and in the presence of added TGF-beta1. Thus, when TGF-beta acts as a co-stimulator for T-cell activation it inhibits the induction of apoptosis and sustains the number of viable cells.

Selective cytotoxicity of two rodent T cell lymphomas to rat yolk sac tumours involves a retroviral envelope protein expressed by the lymphoma.

A Gross virus induced rat T cell lymphoma G1-Tc1 and a Moloney virus induced mouse T cell lymphoma YAC-1 are shown to exert a strong cytotoxic activity against rat yolk sac tumours but not to various types of rat, mouse or human normal cells or tumour cell lines including carcinomas, sarcomas, lymphomas and gliomas. Both lymphomas are CD3+, CD4-, CD8- and T-cell receptor (TCR) alpha beta +. The cytotoxicity was not MHC restricted or dependent on the density of MHC class I of the target cells, and the mouse lymphoma killed the rat yolk sac tumour target. The cytotoxic action was fast and up to 80% specific killing was observed in 4-h 51Cr release assays. A rat B cell hybridoma was established from a Wistar/Furth (WF) rat immunized with the syngeneic lymphoma G1-Tc1 producing an immunoglobulin (Ig)G2c monoclonal antibody (MoAb) 1F2. This binds to the lymphomas G1-Tc1 and YAC-1 and also to a murine non-cytolytic Rauscher lymphoma RMA, but not to any other of several rat, mouse or human cell types tested. The 1F2 completely inhibited the killing of rat yolk sac tumours by the two cytolytic lymphomas, but did not interfere with the killing mediated by natural killer (NK) cells or cytolytic lymphokine-activated killer (LAK) cells. Immunochemical analysis of solubilized cell membranes of the lymphoma G1-Tc1 demonstrates that the 1F2 antibody recognizes an epitope on a retroviral gp 70 envelope protein. This indicates that a retroviral protein is involved in the lytic activity of the two lymphomas.