Regualtion of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts.

A/Jax mice were rendered immune to the syngeneic and transplantable methylcholanthrene-induced Sarcoma 1509a by the surgical removal of the tumor 7 days after implantation; subsequent injection i.v. transfer of 10(7) to 10(8) washed thymus or spleen cells of tumor-bearing animals (TBA) to immune animals significantly inhibited the rejection of the tumor; this suppressive effect was entirely abolished by the treatment of these lymphocytes with anti-theta serum or anti-thymocyte serum (ATS) and complement before adoptive transfer. On the other hand, an equal number of thymus or spleen cells of normal animals or of animals bearing an unrelated tumor had no suppressive effect. Treatment of normal syngeneic animals with ATS after tumor cell inoculation or splenectomy of TBA resulted in the suppression of the tumor growth. The serum of TBA had no effect on tumor growth in immune syngeneic mice. Together these results suggest that TBA possess immunosuppressor T cells regulating negatively their immune response to the tumor.     

Reciprocal T cell responses in the liver and thymus of mice injected with syngeneic tumor cells.

We investigated the T cell responses in various tissues, especially in the liver and thymus, of mice injected with syngeneic tumors. This study was undertaken since recent evidence indicated that the liver is one of the important immune organs for T cell proliferation. When C3H/He mice were intraperitoneally injected with mitomycin-treated syngeneic MH134 tumors (1 x 10(7)/mouse), a transient increase of liver mononuclear cells (MNC) was induced, showing a peak at Day 4 after injection. Histological study of such liver showed a sinusoidal dilatation and an accumulation of MNC in the sinusoids. The most predominant MNC induced were double negative (CD4-8-) alpha beta T cells and gamma delta T cells. These gamma delta T cells varied, showing unique time-kinetics. Despite a continuous increase of whole liver MNC and alpha beta T cells, the proportion of gamma delta T cells in the liver decreased beginning 4 days after injection. In contrast with the response in the liver, a striking decrease in the cell number of thymocytes was induced after tumor injection, showing a basal level at Day 6. This hypocellularity in the thymus appears to be an inverted response of the lymphocytosis in the liver. At this time, a corresponding decrease in the proportion of double positive (CD4+8+) T cells was always seen in the thymus. Analysis of cell proliferative response showed that the increase of liver MNC after tumor injection was accompanied by augmented proliferation, whereas the decrease of thymocytes was accompanied by depressed proliferation. The present results indicate that there exists a unique, reciprocal response of T lymphocytes between the liver and thymus, and that the presence of tumor appears to stimulate T cell response in the liver but alternatively inactivates such response in the thymus.     

Characteristics of the immature cells involved in T cell-mediated enhancement of syngeneic tumor growth.

Enhancement of tumor growth was observed when non-sensitized thymocytes were injected together with tumor cells into syngeneic mice, although this tumor enhancement was less pronounced than that caused by tumor-sensitized T lymphocytes. The cells within the thymus which are responsible for this tumor enhancement were found to be rapidly dividing and to be absent from the thymus a day after cortisone administration. At a longer time interval the cortison-depleted thymus was repopulated by dividing cells which exhibited tumor-enhancing reactivity. The characteristics of these cells suggest that they are in the early stages of thymic processing. The enhancing thymocytes were sensitive to treatment with the thymic humoral factor which functions in T cell maturation, and their enhancing activity was cancelled by such treatment. These results are compatible with our hypothesis that exposure of immature T cells to a tumor stimulus may lead to tumor enhancement whereas interaction between mature T lymphocytes and tumor cells may be required for tumor inhibition.     

Action of embryonic thymus and liver cells on the development of the ascitic form of Ehrlich's carcinoma

The influence of the cells of embryonic thymus and liver on the development of Ehrlich carcinoma was studied. The intraperitoneal injection of the embryonic cells in the adult mice infested by the Ehrlich carcinoma resulted in a marked lengthening of the life time of animals and an increase of the survival percentage. The embryonic cells of thymus and liver inhibited sharply the growth of carcinoma cells in the diffusion chambers as well. In contrast to this, the thymus and bone marrow cells of adult animals, taken in the same concentrations as the embryonic cells, exhibited only a slight inhibiting effect on the growth of tumour cells. On the basis of these data a suggestion is put forward to the effect that the embryonic immunocompetent cells determine the stronger inhibition of tumour growth in the embryos as compared with the adult animals.     

Opposing reactivities of subpopulations of T lymphocytes toward syngeneic tumor cells: separation of early thymocytes.

The present communication is a continuation of earlier studies which indicated that interaction between syngeneic tumors and those lymphocytes in the early stages of thymic processing can result in enhanced tumor growth in vivo. The thymocytes involved in this tumor enhancement were found previously in the rapidly dividing subpopulation of subcapsular cortical thymocytes, both in the untreated thymus and in the thymus undergoing repopulation after cortisone depletion. In the present experiments we have isolated this small subpopulation of early thymocytes. After cortisone injection such cells could be separated from the medullary cortisone-resistant thymocytes since the latter cells exhibit a high level of surface H-2 antigens and were thus lysed preferentially by anti-H-2 serum and complement. The repopulating subcapsular early thymocytes, which were resistant to this treatment, were incapable of responding to PHA while their basal proliferation rate was undiminished, and the majority of the cells were found to be dividing. When such low H-2 early thymocytes were injected together with three different tumors into syngeneic mice their tumor-enhancing activity was evident. It is clear that such early thymocytes are not devoid of biologic reactivity and their release from the thymus could have decisive results.     

Release of immature cells from the thymus during solid tumor growth: identification by assay of TdT activity.

In vivo anti-tumor activity of spleen cells from C3H/eb mice bearing a syngeneic fibrosarcoma was shown previously to decline to an undetectable level and be replaced by tumor-enhancing activity as tumor growth proceeds. In the light of our findings that thymocytes in the early stages of thymic processing can bring about tumor enhancement, we postulated that premature release of thymocytes and their accumulation in the spleen might account for the loss of the anti-tumor response. In the present experiments an injection of thymocytes did in fact cancel the anti-tumor response of reactive splenocytes from tumor-bearing mice. In order to determine whether premature thymocyte release occurs naturally in the tumor-bearing animals, we assayed activity of the enzyme TdT (as a marker for thymus cells) in the spleens of these mice during progressive tumor growth. Cells with TdT activity were clearly evident in the spleens of the tumor-bearing animals, were derived from the thymus, and accumulated in parallel to the loss of anti-tumour reactivity.     

Immunologic suppression of carcinogenesis in spontaneously hypertensive rats (SHR) with T cell depression

A strain of spontaneously hypertensive rats (SHR) showed a selective depression of T cell functions brought about by aging. Conversely, this strain had a high NK cell activity as compared to other normal rat strains. This SHR strain was found to be much more sensitive to the carcinogenic activity of low doses of MCA than were WKA rats with normal T cell functions. Allogeneic thymus grafts almost completely restored the T cell functions of SHR, whereas injection of an immunopotentiator, NSP, enhanced NK cell activity and also caused a partial recovering of T cell functions. When immunologic restoration was achieved, generation of killer T cells to syngeneic SMT-5 tumor cells was induced and the cytotoxic activity of NK cells to K-562 cells was also enhanced. But the cytotoxic activity to the SMT-5 cells of NK cells and macrophages from the treated or untreated SHR was not detected. Allogeneic thymus grafts induced a significant transplantation resistance against a syngeneic SMT-5 tumor and injection of NSP enhanced only the survival days of the rats. Allogeneic thymus grafts also significantly suppressed the incidence of tumors induced by MCA, whereas the injection of NSP was not effective in the prevention of tumor development but was effective in prolongation of latency periods. These results support the hypothesis that immune surveillance mediated by T cells is an important mechanism for the control of tumor development.     

Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte.

The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately 10(4) fold by the addition of 2 X 10(6) normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG8 myeloma tumor cells secreting IgG1. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth-supporting factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cell abolishes their growth-enhancing properties. Thymus cells can be syngeneic and allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-8 week old C3H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of XB3-AG8 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shown that these B cells divide every 18 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immonoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.     

Protective effects of diosmetin extracted from Galium verum L. on the thymus of U14-bearing mice

Diosmetin (DGVL) extracted from the traditional Chinese herb Galium verum L. has been found to have anticancer activity. In this study, the effects of DGVL on the thymus of U14-bearing mice were investigated. Using flow cytometry, peripheral blood lymphocytes were characterized based on the expression of surface markers for T helper cells (CD4(+)) and T suppressor cells (CD8(+)). Serum levels of tumor necrosis factor α (TNF-α), interleukin-2 (IL-2), IL-10, and transforming growth factor β1 (TGF-β1) and a cell proliferation assay were determined with an enzyme-linked immunosorbent assay. The expression of Fas and Fas ligand (FasL) on the thymus was determined by Western blotting. Our results showed that DGVL inhibited tumor growth and significantly increased the thymus weight compared with the control. Also, DGVL elevated serum levels of IL-2 and significantly reduced levels of TNF-α, TGF-β1, and IL-10 in a dose-dependent manner. Histological study and terminal dUTP nick end labeling staining results showed that DGVL protected thymus tissue against the onslaught of tumor growth by inhibiting thymus lymphocyte apoptosis. The cell proliferation assay revealed that DGVL might promote more thymus lymphocytes towards proliferation. Furthermore, the ratio of CD4(+)/CD8(+) T lymphocytes was significantly increased from 0.69 to 2.29 by treatment with DGVL. Immunoblotting analyses revealed that the expression of Fas and FasL on the thymus was lower in mice in the DGVL treatment group than in the control mice. In conclusion, DGVL can inhibit tumor growth and protect tumor-induced apoptosis of the thymus, and the mechanism is closely associated with reduced cell death in the thymus and a Fas-FasL-dependent pathway.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

The inhibition by the tumor necrosis factor of the death and DNA fragmentation of lymphoid cells in irradiated rats]

The influence of a tumor necrosis factor, administered 16 h before irradiation of rats, on the radiation response of thymus and bone marrow cells has been investigated. Three and 6 h after irradiation the following indices were analyzed: the number of apoptotic cells in the thymus; the accumulation of polydeoxyribonucleotides and the appearance of single-strand breaks in DNA of bone marrow and thymus cells; and the electrophoretic properties of thymocyte DNA. The injection of a tumor necrosis factor reduced the number of polydeoxyribonucleotides, inhibited internucleosome DNA fragmentation, and did not influence the formation of single-strand breaks in DNA.     

Angiogenesis gene expression profiling in xenograft models to study cellular interactions

The present study describes a method to simultaneously obtain the angiogenic expression profile in tumor cells and vascular cells of a single tumor. Human- and mouse-specific primers were used for quantitative real-time RT-PCR to determine the expression of vascular endothelial growth factors A, B, C, and D, vascular endothelial growth factor receptors 1, 2, and 3, neuropilin 1 and 2, angiopoietin 1, 2, 3/4, tyrosine kinase receptors 1 and 2, basic fibroblast growth factor (bFGF) in xenograft tumors obtained by injection of human ovarian carcinoma cells in nude mice. In addition, the effect of treatment with anginex and taxol on the expression profile was analyzed. Most factors were expressed higher in vascular cells as compared to tumor cells. In response to treatment, tumor cells significantly upregulated bFGF expression and downregulated VEGF receptor expression. This was accompanied by downregulation of VEGF-B and -D, and upregulation of angiopoietin-3 as well as angiopoetin receptors in nontumor cells. In conclusion, real-time qRT-PCR combined with xenograft tumor models presents a sensitive method to monitor angiogenesis and to analyze interactions between tumor cells and nontumor cells in vivo. The approach can be applied to different research fields in which xenograft models are used.     

Prevention of macrophage tumoricidal activity by agents known to increase cellular cyclic AMP.

The concept of T-T cell interaction which was first suggested during cell-mediated immune response to alloantigens was evaluated in a syngeneic tumor system. The combination of lymph node and thymus cells from BALB/c mice immune against syngeneic tumor cells, mKSA, was shown to exhibit collaboration with respect to in vitro generation of effector cells capable of preventing growth of corresponding tumor cells in the tumor cell neutralization assay. While each cell population of either anatomical site did not prevent tumor growth when tested alone, combinations of both did. The antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding lymph node cells. The lymph node and thymus cell populations participating in synergy were found to be thymus dependent. These results suggest that we may be dealing with the same or similar T₁- and T₂-cell populations described before as displaying synergy in response to alloantigens in the graft versus host, mixed lymphocyte, and cell-mediated cytotoxicity reactions.     

Thymus cell population exerting a regulatory function in the immune response of mice to polyvinyl pyrrolidone

An increased response to PVP was observed after adult thymectomy and was partially reversed either by thymus implantation or by a single injection of thymic cells. In addition, an injection of thymic cells was found to reduce the response to PVP in normal recipients. An enhanced response to PVP was measured in B mice compared to that of normals. In such mice reduction of the response to PVP was observed when repeated doses of thymus cells were administered. Lower doses of HC resistant thymus cells strongly inhibited the response to PVP. The cells involved in the thymus regulatory function appear to be radiosensitive, since it was shown that radiation by itself resulted in an increased response to PVP. This inhibitory function of the thymus seems to disappear relatively early in progression of life, as seen by an increased response to PVP in elder mice. These results indicate that a T cell population exerts a regulatory function in the immunological response to PVP that was previously considered to be thymus independent.     

An intradermal assay for quantification and kinetics studies of tumor angiogenesis in mice.

A method is reported for the study of early phases of neovascularization in syngeneic murine tumors and human tumor xenografts in nude mice. Using this method, the effect of irradiation of tumor cells or tumor bed on tumor angiogenesis was studied. Tumor cells were injected intradermally in the abdominal skin flap, which was reopened at 2-day intervals to quantify newly formed blood vessels at the site of tumor cell injection. Both tumor cell injection and blood vessel counting were performed under a dissecting microscope. Using three syngeneic murine tumors and two clones of a human colonic adenocarcinoma, it was observed that new blood vessels started appearing within a few days after tumor cell injection and that this event preceded measurable tumor growth. The number of blood vessels increased exponentially for several days but then their further growth slowed. The extent of angiogenesis depended on the tumor type and the number of tumor cells injected. The exposure of the skin flap to ionizing radiation prior to tumor cell injection reduced neovascularization. We further observed that heavily irradiated tumor cells retained their ability to induce angiogenic response and that lymphoid cells (peritoneal exudate and spleen cells) could also elicit an angiogenic response, although it is weaker than the response elicited by tumor cells. Thus this method is suitable for quantification and kinetics of early phases of tumor angiogenesis in individual mice bearing transplants of syngeneic tumors or human tumor xenografts, and it can be useful for investigating various regulators of tumor angiogenesis.     

Cell cooperation in abolition of tolerance of xenogeneic tumor.

Thymectomized, lethally irradiated mice reconstituted with syngeneic bone marrow cells are tolerant to xenogeneic Yoshida ascites sarcoma (YAS). The tolerance was abolished by an injection of syngeneic normal spleen, thymus, or lymph node cells given simultaneously with YAS. Allogeneic and semiallogeneic spleen cells were ineffective. The YAS-rejecting mice produced specific anti-tumor antibodies. The serum of these mice transferred to tolerant T-cell-deficient mice protected the latter from inoculated YAS cells. These serum-protected mice were not able to resist the reinoculum of the tumor cells as the mice restored with lymphoid cells did. The latter mice rejected the YAS at the time when donor cells were practically absent in their lymphoid tissue. The low effective ratio of injected syngeneic lymphoid to tumor cells, efficiency of injected thymus cells, and other data led to the conclusion that transferred lymphoid cells did not act directly on tumor cells but through cooperation with host lymphoid cells. The cooperation of donor T- and host B-lymphocytes enabled the activation of the latter, and YAS cells were rejected.     

T cell recruitment from the thymus to the spleen in tumor-bearing mice

Summary After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1⁺ cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE₂ levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE₂ level by replenishment with exogenous PGE₂ inhibited the splenic cell increase in tumor bearers. In the tumorbearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE₂ in the presence of a tumor.Abbreviations PNA⁺ cells, peanut agglutinin binding cells - Ig⁺ cells, surface IgM positive cells - Thy 1⁺ Thy 1.2 antigen positive cells - PGE₂ prostaglandin E₂     

Dihydroartemisinin shift the immune response towards Th1, inhibit the tumor growth in vitro and in vivo

Some investigators have been found that Artemisinin and its derivates have inhibitory effect on growth of cancer cells. Among these derivatives, Dihydroartemisinin (DHA) is well known as a semi-synthetic one. In addition, T cells are proved to be essential for the destruction of cancer cells. In this research, we assessed the effects of DHA on tumor cell growth inhibition in vitro by MTT assay and in vivo by intra tumor injection of DHA against breast cancer. The results showed that the IC₅₀ values of DHA for RIN pancreatic tumor cell line were 30 μM and significant decrease in the tumor size in vivo. Also we evaluate the effect of DHA on the modulation of immune response in tumor bearing animals; these include the splenocyte proliferation using the BrdU kit; measurement of cytokine profile by ELISA, and evaluate the percentage of T regulatory cells in the spleen by flowcytometry. Our results demonstrated that a significant decrease in the level of IL-4 in the animals treated with DHA and significant decreased in the level of splenic CD4⁺CD25⁺ Foxp3⁺ T regulatory cells.     

Tumor immunity in the mammary gland

Summary Mouse mammary tumor 410, which was derived from a spontaneously arising BALB/cf C3H mammary tumor, grows better in syngeneic BALB/c mice after injection into mammary fatpads than after injection into subcutaneous sites. This finding is consistent with the notion that the fatpad is an imunologically privileged site. However, no evidence that the mammary fatpad was immunologically privileged with respect to tumor transplantation antigens was found. Tumor cells were injected into mammary fatpads or SC. When the tumors became palpable they were surgically removed. One to three weeks later, the mice were challenged on the opposite side by injection of tumor cells either SC or into the mammary fatpad. The mice were immune after temporary growth of tumors either in the fatpad or SC. Regardless of the growth site of the immunizing tumor, the mice rejected the challenge tumor cells whether they were injected SC or into the fatpad.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.