Hans Olov Sjögren

Biosynthesis of corticotropin-releasing hormone in human T-lymphocytes

Corticotropin-releasing hormone (CRH) is a 41-amino acid neuropeptide which increases the transcription of the proopiomelanocortin (POMC) gene, as well as the biosynthesis and secretion of POMC-derived peptides. Using a specific human CRH radioimmunoassay we have shown that human T-lymphocytes contain immunoreactive CRH. We studied the effects of phytohemagglutinin (PHA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the biosynthesis of CRH in human T-lymphocyte cell cultures. A significant increase in CRH mRNA levels was observed in human lymphocytes after 12 h of PHA/TPA treatment, while the levels decreased after 22 h. These findings could imply an immunomodulatory role for CRH that could be due to autocrine and/or paracrine interactions.

Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-γ, 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-γ (IFN-γ), 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-γ and IL-7, respectively. The strongest effect was observed with IFN-γ-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.

Cure of established, intracerebral rat gliomas induced by therapeutic immunizations with tumor cells and purified APC or adjuvant IFN-gamma treatment.

We have previously reported that immunizations with mutagen-induced immunogenic variants of a weakly immunogenic rat glioma could protect against isografts of the original tumor cells. In this study we show that prolonged survival and cures of rats with established gliomas in their brains can be achieved by therapeutic immunizations with tumor cell mutants, combined with in vitro and in vivo interferon (IFN)-gamma (adjuvant) treatment, or tumor cells admixed with semipurified syngeneic dendritic cells. Cure of rats with established intracerebral gliomas was possible when immunizations were initiated up to 5 days after intracerebral isografting of original tumor cells. Unexpectedly, immunizations combined with in vitro and in vivo IFN-gamma treatment or with admixed semipurified dendritic cells equalized the immunogenic potential of the original tumor cells and that of mutagen-induced immunogenic cell variants (tum-). This demonstrates that effective immunizations against a weakly immunogenic brain tumor can be achieved by different adjuvant concepts. The therapeutic effect of immunizations with tumor cells admixed with semipurified dendritic cells was highly significant in female rats, whereas only occasional cures and prolonged survival were recorded in male rats. The overall results show that therapeutic immunizations can indeed be effective against an established and growing intracerebral tumor.

Immunization with mutagen-treated (tum-) cells causes rejection of nonimmunogenic rat glioma isografts.

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compoundN-methyl-N′-nitro-N-nitrosoguanidine and the surviving cells cloned by limiting dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum−) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon γ (IFNγ) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum− clones pretreated with IFNγ induced significant rejection against intracerebral N32 isografts. Both N32 and its tum− closes were MHC class I positive and MHC class II negative. IFNγ treatment enhanced the MHC class I expression with 20%–90% on the tum− clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFNγ treatment but not on any of the tum− clones tested. We conclude that the enhancing effect of IFNγ treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum− variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.

A glioma classification scheme based on coexpression modules of EGFR and PDGFRA

Significance Classification of cancer provides crucial guidance for clinical treatment and mechanistic studies. Our work extends previous glioma classification studies in that we established EGFR module (EM)/PDGFRA module (PM) glioma classification scheme based on gene coexpression modules around key signaling pathways conserved in neural development and gliomagenesis. We identified coexpressed EM and PM genes as classifiers. Based on the EM and PM signatures, our classification scheme robustly assigns adult low-grade and high-grade diffuse gliomas into three major subtypes that are distinct in patient survival, and in transcriptomic and genomic patterns. Our work suggests that EM and PM genes may play currently unrecognized roles in gliomagenesis. EM/PM glioma classification scheme forms a framework toward establishing molecular diagnostic tools and identifying new therapeutic targets to combat gliomas. We hypothesized that key signaling pathways of glioma genesis might enable the molecular classification of gliomas. Gene coexpression modules around epidermal growth factor receptor (EGFR) (EM, 29 genes) or platelet derived growth factor receptor A (PDGFRA) (PM, 40 genes) in gliomas were identified. Based on EM and PM expression signatures, nonnegative matrix factorization reproducibly clustered 1,369 adult diffuse gliomas WHO grades II-IV from four independent databases generated in three continents, into the subtypes (EM, PM and EMlowPMlow gliomas) in a morphology-independent manner. Besides their distinct patterns of genomic alterations, EM gliomas were associated with higher age at diagnosis, poorer prognosis, and stronger expression of neural stem cell and astrogenesis genes. Both PM and EMlowPMlow gliomas were associated with younger age at diagnosis and better prognosis. PM gliomas were enriched in the expression of oligodendrogenesis genes, whereas EMlowPMlow gliomas were enriched in the signatures of mature neurons and oligodendrocytes. The EM/PM-based molecular classification scheme is applicable to adult low-grade and high-grade diffuse gliomas, and outperforms existing classification schemes in assigning diffuse gliomas to subtypes with distinct transcriptomic and genomic profiles. The majority of the EM/PM classifiers, including regulators of glial fate decisions, have not been extensively studied in glioma biology. Subsets of these classifiers were coexpressed in mouse glial precursor cells, and frequently amplified or lost in an EM/PM glioma subtype-specific manner, resulting in somatic copy number alteration-dependent gene expression that contributes to EM/PM signatures in glioma samples. EM/PM-based molecular classification provides a molecular diagnostic framework to expedite the search for new glioma therapeutic targets.

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

Abstract. 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 intra-hepatic colon carcinoma, H1D2, the spleen cell anti-tumor 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-γ 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.

Fractionation of rat IgG subclasses and screening for IgG Fc-binding to bacteria

The four IgG subclasses of the rat, IgGl, IgG2a, IgG2b and IgG2c, were purified from normal serum by a combination of protein A-affinity chromatography and DEAE-cellulose chromatography. Purified, radiolabelled preparations of IgG were tested for binding to Gram-positive bacteria representing five different Fc-receptor (FcR) types. Distinct rat subclass-specific Fc-binding was noted to bacterial species belonging to different Fc-receptor types. Staphylococcus aureus (FcR I) strains bind IgGl and IgG2c as shown by others. Group C and G Streptococci (FcR III) bind all four subclasses of rat IgG. Streptococcus zooepidemicus strains (FcR V) also bind all four subclases but only to a lower degree. Human group A Streptococci (FcR II) and bovine group G Streptococci (FcR IV) do not bind any of the rat IgG subclasses. Elution studies on two strains. Staphylococcus aureus, Cowan I, and human group G Streptococcus, G 148, showed that both thiocyanate and pH-elution might be useful for the fractionation of IgG subclasses bound to bacterial cells. The present work indicates the possible use of bacterial cells as solid-phase absorbents in immunological studies of rat IgG.

Low-Dose Combretastatin A4 Phosphate Enhances the Immune Response of Tumor Hosts to Experimental Colon Carcinoma

Purpose: Although there is a need to enhance the therapeutic efficiency in cancer by combining immunotherapeutic procedures with other therapy, combination with chemotherapy is complicated due to immunosuppressive effects of most chemotherapeutic drugs. The purpose of this investigation was to study whether combining tumor cell immunization with the vascular targeting drug combretastatin A4 phosphate (CA4P) would enhance tumor retardation and/or affect the antitumor immune response. Experimental Design: Rats with intrahepatic colon carcinoma were immunized weekly with IL-18/IFNγ–transfected tumor cells, starting day 9, and were treated with a low-dose CA4P (2 mg/kg, 5 days a week starting day 7). The effect of CA4P was studied on tumor growth and on immune reactivity in vitro. Results: Rats with preexisting tumor, immunized and treated with low-dose CA4P, had a significantly retarded tumor growth compared with rats receiving CA4P or immunization alone. Splenocytes from rats treated with this combination had a significantly enhanced antitumor immune response compared with splenocytes from control rats. Exposure of nonadherent splenocytes to CA4P in vitro did not enhance their proliferation. However, 3-hour pretreatment of adherent splenocytes with 0.3 μg/mL CA4P significantly enhanced proliferation and IFNγ production of admixed nonadherent splenocytes, partly due to nitric oxide reduction. Combining the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester with CA4P and immunization further retarded tumor growth. Conclusion: Concomitant treatment of rats with progressively growing tumor with immunization and low-dose CA4P significantly enhances the therapeutic effect as compared with either treatment alone and results in an enhanced antitumor immune reactivity.

Influence in vitro on NK and K cell activities by cimetidine and indomethacin with and without simultaneous exposure to interferon

SummaryThe histamine-2 receptor antagonist cimetidine (10−5 M) and the prostaglandin synthesis inhibitor indomethacin (10−8 M) augmented natural killer cell activity in the majority of healthy controls and patients with advanced melanoma and in a lower frequency of patients with colorectal carcinoma. Antibody-dependent cellular cytotoxicity was increased in most melanoma patients but in a lower proportion of patients with colorectal cancer. Compared with the effect of interferon the augmentation of NK- and K-cell activities was small in most patients. Cimetidine was also demonstrated to bring about a further increase in the interferon-induced NK activation of peripheral blood mononuclear cells from a majority of healthy donors and patients with melanoma. Furthermore, cimetidine augmented the interferon-induced K-cell activation of peripheral blood mononuclear cells from most patients with melanoma and colorectal cancer.

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.