Recognition by cellular and humoral autologous immunity in a human osteosarcoma cell line

Because of the difficulty of developing pairs of osteosarcoma cell lines and cytotoxic T lymphocytes (CTLs), no osteosarcoma tumor antigens that are useful for antiosteosarcoma immunotherapy have yet been identified. In parallel with continuous attempts to develop such pairs from osteosarcoma, we employed serological identification using a recombinant expression cloning (SEREX) method to identify B cell-defined antigens. Consequently, a human osteosarcoma cell line, OS2000, was established from a primary osteosarcoma of a patient cured of hereditary retinoblastoma. Repetitious in vitro stimulations by OS2000 cells to the autologous peripheral T cells induced cytotoxic activity in the autologous osteosarcoma cells but not in the nontumor cells. The cytotoxicity was inhibited by anti-HLA class I monoclonal antibody. SEREX analysis revealed that autologous humoral immunity reacted to two proteins expressed in OS2000. One was the self HLA-Cw*0102 molecule, and the other was wild-type smooth muscle myosin light chain (SMMLC). However, no antigenicity of these proteins was seen versus the sera of the other patients. In conclusion, our results demonstrated the presence of host cellular and humoral immune responses to autologous osteosarcoma cells. This offered the opportunity to identify osteosarcoma antigens recognized by autologous immunity.     

Glioma immunotherapy with combined autologous tumor cell and endothelial cell vaccine in vivo

Combined vaccines containing GL261 murine glioma cells and F-2 murine endothelial cells fixed with glutaraldehyde-phosphate buffered saline were injected into the intradermal tissue of the tail base of C57BL/6 mice. After the vaccination, GL261 cells were injected subcutaneously into the left flank of the mice. Vaccination with fixed F-2 cells induced the development of relatively high amounts of interferon-gamma-releasing cells after in vitro re-stimulation with vascular endothelial growth factor-receptor 2 peptide. Tumor growth was inhibited after preventive use of the combined vaccine, prepared from GL261 and F-2 cells. Tumor specimens obtained from the combined vaccine group in a therapeutic experiment showed significantly decreased vessel count. Glioma immunotherapy with a combined vaccine prepared from tumor cells and endothelial cells might represent a new clinical strategy, as such combinations may theoretically affect both high-grade glioma cells and their environment.     

Cytotoxicity of lymphocytes sensitized by autologous tumor cells in vitro, against autologous lung cancer cell lines

In order to obtain killer lymphocytes that would have a strong cytotoxicity against autologous tumor cells, we conducted a cytotoxic assay of effector cells cultured from human peripheral blood lymphocytes (PBL) using 10 cultured lung cancer cell lines as target cells. We present this result. (1) Lymphocytes obtained by 17 days mixed lymphocyte tumor cell culture (MLTC: Fresh PBL from lung cancer patients was mixed with irradiated autologous tumor cells in a medium, and cultured for 3 days. Irradiated allogeneic lymphocytes were then added, and cultured for 14 days) were cytotoxic against 4 of the 10 autologous lung cancer cell lines. (2) Lymphocytes obtained by 14 days MLTC and 3 more days culture in a medium containing interleukin 2 were cytotoxic against all 10 of the autologous lung cancer cell lines. These lymphocytes proliferated to 4.13 times of the original cell number, and their surface marker was OKT3+. These killer lymphocytes are expected to be effective in adoptive immunotherapy as effector cells.     

Suppression of primary tumor growth in a mouse model of human neuroblastoma

BACKGROUND/PURPOSE: Neuroblastoma is the most common tumor of the abdomen in children. Consistently effective treatments are lacking for aggressive disease. The authors previously reported that therapy with anti-vascular endothelial growth factor (VEGF) antibodies suppresses both growth and metastasis in an experimental model of Wilms' tumor. The authors hypothesized that, in a parallel model of neuroblastoma, anti-VEGF treatment would inhibit (1) growth and (2) metastasis. METHODS: Primary tumors were established in the kidneys of nude mice. In cohort 1 (n = 42), mice were killed at 3 time-points, and tissues were evaluated histologically. Tumors were assayed for VEGF. In cohort 2 (n = 28), anti-VEGF antibody or vehicle was administered. Tumor weights and the incidence of metastases in the 2 groups were compared. VEGF deposition was evaluated by immunohistochemistry. RESULTS: Mice displayed large tumors with liver and lung metastases. VEGF levels in tumors increased over time. Antibody-treated animals displayed significantly smaller tumors, but incidence and size of metastases were unaffected. VEGF was localized to tumor stroma immunohistochemically, with no difference in pattern observed in control and antibody-treated tumors. CONCLUSIONS: Anti-VEGF antibodies inhibit primary tumor growth in experimental neuroblastoma, but not metastasis. This may contrast with the effect of the same antibody in a parallel model of Wilms' tumor.     

高剂量化疗合并自体造血干细胞移植治疗睾丸肿瘤

报告高剂量化疗＋自体造血干细胞移植（ＨＤＣ＋ＡＨＳＣＴ）治疗４例睾丸非精原细胞瘤（ＮＳＧＣＴ）的结果。预处理方案由环磷酰胺（３．０～３．５ｇ／Ｍ２）、足叶乙甙（７００～１０００ｍｇ／Ｍ２）、卡铂（６００～７５０ｍｇ／Ｍ２）组成。治疗中骨髓抑制均达Ⅳ度，无Ⅳ度髓外毒性出现。３例初治进展期患者治疗后１例完全缓解，２例部分缓解，其中１例在移植后６个月病情进展，另２例已持续缓解１５个月；１例复发患者治疗后肿瘤进展。认为ＨＤＣ＋ＡＨＳＣＴ治疗ＮＳＧＣＴ的适应证仍有待进一步探索，使用的预处理方案的剂量强度尚可进一步提高     

Suppressor activity of the human spleen and thymus.

Both the spleen and thymus of man contain a population of cells which can suppress the mixed lymphocyte reaction. Splenic cells suppress the MLC 58 +/- 4.8 percent, and the thymus is able to suppress the MLC 90 +/- 2.6 percent. However, splenic cells require stimulation by vegetable mitogens before suppressor activity can be observed, and the thymus displays spontaneous suppressor activity without prior stimulation. The suppressor effect is linearly related to the log of the cell dose (r = 0.8, p less than 0.01), and at low doses the suppressor cells stimulate rather than inhibit the MLC. The cells are exquisitely sensitive to immunosuppressive drugs, and blood levels encountered in clinical organ transplantation abrogate their suppressive activity. The suppressive activity of the cells is nonspecific, and they are able to inhibit the MLC between individuals totally unrelated to the spleen or thymus donor. Since these cells likely play a role in the development of tolerance, further characterization of their properties is required.     

Serologic analysis of human tumor antigens. II. Reactivity of sera from eleven osetogenic sarcoma patients against autologous and allogeneic tumor cells.

Sera from eleven patients bearing osteogenic sarcomas were tested for reactivity against autologous and allogeneic normal and tumor cells using a sensitive microcytotoxicity assay. Following absorption with human fetal tissue cultured lines, sera from all 11 patients lost reactivity to autologous and allogeneic normal fibroblasts in culture. However, sera from four patients retained reactivity against both autologous and allogeneic osteogenic sarcoma, sera from five patients retained reactivity to allogeneic but not autologous tumor, and sera from two patients lost all reactivity to tumor. Sera from nine normal individuals did not react with either normal or osteogenic sarcoma cells after fetal cell absorption. Thus it appears that nine of 11 patients bearing osteogenic sarcoma contained antibody to a common tumor-associated antigen. This tumor-associated antigen appears to be expressed on tumor cells and not normal cultured cells, and reactivity to it is present in tumor-bearing but not normal individuals.     

Active immunotherapy of stage IV renal cell cancer using autologous tumor cells

Summary A total of 53 patients with stage IV renal cell carcinoma were treated by vaccination with autologous tumor cells in Candida-antigen after palliative tumor nephrectomy. Follow-up has been up to 9 years. Complete remission within 48 months after nephrectomy was observed in 3 patients, while 6 showed partial remission and 18 are stable with disease. Of 26 patients with rapid progression, 17 died within 1 year after operation. The best response was seen in metastases to the liver and lung. CNS-lesions or bone metastases do not appear to respond to this treatment. We conclude that this mode of therapy is beneficiary to a certain group of patients and should be offered, as no severe side effects have been observed so far. Whether new perspectives of adoptive immunotherapy using tumor-specific monoclonal antibodies or recombinant interferons will prove effective remains to be seen.     

Suppression of cellular immunity by surgical stress

BACKGROUND: Suppression of cellular immunity is one of the host responses to surgical stress. In cancer patients this immunosuppression may accelerate the growth and metastasis of residual cancer cells, so it is desirable to restrict immunosuppression by surgical stress to a minimum. However, the extent and duration of immunosuppression caused by operations on gastrointestinal cancer, as well as the mechanisms involved, have not been determined. METHODS: To clarify these points, we investigated immunocyte function and measured the blood levels of hormones, cytokines, and acute phase reactants from before to after operation in 20 patients with stage I gastrointestinal cancer. RESULTS: In patients exposed to surgical stress, peripheral blood lymphocyte numbers and function were suppressed until at least 2 weeks postoperatively. This immunosuppression was mainly due to a decrease of helper-inducer T cells, cytotoxic T cells, natural killer cells, and interleukin-2 receptor-positive cells, as well as an increase of suppressor T cells. In addition, hypersecretion of cortisol and overproduction of immunosuppressive acidic protein were observed. CONCLUSIONS: Cellular immunosuppression by surgical stress was mainly due to an increase of lymphocyte subsets that depress cellular immunity coupled with a decrease of the subsets that promote it. Overproduction of cortisol and immunosuppressive acidic protein in response to surgical stress may play an important role in the development of immunosuppression.     

Suppression of antigraft immunity by preimmunization. I. Kinetic aspects and specificity

Intravenous injection of 2,000 rad of irradiated allogeneic cells can suppress the development of antigraft delayed-type hypersensitivity (DTH) to major and minor histocompatibility (H) antigens which normally arises after s.c. immunization. Secondary type DTH responses to minor H antigens were also largely suppressed by an i.v. injection of irradiated allogeneic cells 1 week preceding the s.c. priming injection. The extent of suppression of primary DTH to allogeneic H-2-incompatible cells depended on the dose of i.v. injected irradiated cells. After a dose of 1 x 10(7) irradiated spleen cells i.v., the suppression persisted for at least 40 days. Intravenous injection of cells incompatible for minor H antigens could not suppress the DTH to H-2 alloantigens and vice versa. Suppression of DTH to H-2 alloantigens was haplotype specific. Proliferation studies indicated that the immunosuppressed mice do not respond upon s.c. immunization with an increased proliferative activity in the draining lymph nodes, in contrast to nonsuppressed mice. The data suggest that i.v. preimmunization with allogeneic cells induces specific suppression of antigraft immunity acting at the induction stage of the immune response.     

Suppression of murine immunologic functions by fresh and cultured human tumor extracts

The role of human tumor-derived immunoregulatory factors (IRF) in the suppression of murine in vitro cell-mediated immune systems was investigated. IRF was extracted from a fresh human colon carcinoma and a liposarcoma using 3 M KCl. These extracts have previously been shown to suppress in vitro human immune responses. Both IRF extracts inhibited PHA-stimulated murine splenocyte [3H]Tdr uptake in a dose-dependent manner while extracts of normal tissue were not inhibitory. To further investigate in vitro immunosuppression a (C57BL/6 X A/J) F1 anti-B10. BR mixed lymphocyte reaction (MLR) was developed. Optimal [3H]Tdr incorporation was on Day 4 with 1 X 10(5) responders and 2 X 10(5) irradiated stimulators. Addition of IRF caused a 56% inhibition of this response but did not alter the kinetics of the MLR response. Induction of cell-mediated cytotoxicity (C57BL/6 X A/J F1 vs B10.D2) was significantly inhibited by addition of IRF during in vitro sensitization. Release of 51Cr from P-815 targets was decreased to spontaneous release levels at an effector:target (E:T) ratio of 20:1 when IRF was present during sensitization. At this E:T ratio, cells sensitized in the presence of a normal muscle 3 M KCl extract or medium caused 71 and 60% 51Cr release, respectively. IRF activity could also reproducibly be extracted from two small cell lung carcinoma tissue culture lines grown under a variety of culture conditions or passaged in nu/nu mice. The biochemical characteristics of the factor inhibiting human and murine lymphoid cell proliferation were identical. Thus, this system provides a convenient model for assessing the activity of human tumor-derived immunoregulatory factors.     

Suppression of antigraft immunity by preimmunization. II. Characterization of the suppressor cells

Immunization of mice with irradiated (20 Gy) or non-irradiated allogeneic spleen cells i.v. induces delayed-type hypersensitivity (DTH)-reactive T cells, as well as suppressor T cells, against histocompatibility antigens. The suppressor T cells are unable to suppress the induction and functional activity of the simultaneously activated DTH-reactive T cells. However, the suppressor T cells do suppress the generation of DTH-reactive T cells after subsequent s.c. immunization of the same mice, and after transfer into secondary recipients. Systemic transfer of suppressor T cells is effective the first few days after their induction, and affects the afferent limb of the DTH response. The population of suppressor T cells, which is essential for the systemic transfer of suppression, appeared to be Lyt-1+2+. Splenectomy experiments showed that the spleen is not essential for induction of the suppressor T cells. The precursors of the suppressor T cells belong to the pool of recirculating T lymphocytes; they are insensitive to adult thymectomy and can be depleted by antithymocyte serum treatment.