Purification and characterization of an immunosuppressive factor from ovarian cancer ascites fluid

The effect of cell-free ascitic fluid from patients with epithelial ovarian carcinoma on the generation of lymphokine-activated killer cells (LAK) was compared to the activity generated in control medium containing 10% fetal bovine serum, using Daudi target cells. Samples of ascitic fluid from nine different patients tested inhibited LAK generation. Suppressive activity was evident as early as 24 h of incubation in the presence of ascitic fluid and increasing suppression developed with prolonged exposure. Suppression was concentration-dependent, present at 10%-20% and increasing with concentrations up to 80%. The suppressive effect of ascitic fluid was only partially reversed on increasing the concentration of interleukin-2 (IL-2) from 10 units to 1000 units/ml. Activated LAK appeared to maintain the majority of their activity on further culture in ascitic fluid in the presence of IL-2 but further enhancement of lytic activity was prevented. Fractionation of a suppressive sample by HPLC, using 0.1 M KCl/acetic acid buffer pH 2.6, revealed that the dominant peak of suppressive activity eluted at 25 kDa; with pH 7.0 TRIS-buffered saline, most of the activity was lost on the column. Antibody neutralization studies of the 25-kDa suppressive peak as well as on whole ascitic fluid have revealed that transforming growth factor beta (TGF beta) is the major suppressive factor present in ascitic fluid. Factors that suppress LAK generation in vitro were present in all samples tested. The effect on the lytic activity of activated LAK cells was minimal. This suggests that, in the clinical setting, the greatest impact would be achieved by activating LAK cells ex vivo and subsequently transferring them to the peritoneal cavity in the presence of IL-2 rather than by attempting to generate them in situ by injecting IL-2 into the peritoneal cavity. However, reversal of TGF beta-mediated suppression in situ may be necessary to allow local proliferation of LAK cells to achieve an effective killer-to-target ratio.

Section: Discussionmentioning

confidence: 98%

Generation of lymphokine-activated killer cells in human ovarian carcinoma ascitic fluid: Identification of transforming growth factor-β as a suppressive factor

Hirte

Clark

1991

“…In order to avert this effect, purification of immunoprotective TSTA free of tumor-enhancing substances was achieved by pIEF of 3 M KC1 extracts to separate the active material from acidic fractions which facilitate tumor growth in vivo by activating suppressor cells [10,26]. Similar acidic immunosuppressive factors have been described for human cancers [10,18,33]. In contrast to 3 M KC1 extracts, butanol extracts and electrofocused fractions of butanol extracts do not enhance tumor growth [13].…”

Section: High-performance Liquid Chromatographic Analysis Of Pitp-purmentioning

confidence: 99%

The effect of purification on the immunogenicity of tumor-specific transplantation antigens

Saunders

Kahan

Pellis

1985

Immunization with the tumor-specific transplantation antigens (TSTA) of experimental, chemically induced sarcomas engenders specific host resistance to challenge with viable, homotypic neoplastic cells. The strength of tumor resistance depends upon the physical state of the TSTA used for immunization. Treatment with 10(5)-10(6) irradiated tumor cells, a 2-log dose range, induces complete rejection of neoplastic challenges, while immunization within a 1-log dose range with crude 3 M KCl or with 2.5% butanol extracts containing TSTA evokes a weak state of resistance characterized by decreased outgrowth of tumor challenges, but not neoplastic regression. The reduced immunogenicity may be due to either contamination with substances that antagonize host resistance, for example by induction of suppressor cells, or an intrinsic limitation by virtue of the molecular properties of extracted compared with cell-surface TSTA. MCA-F and MCA-D, two noncross-reactive fibrosarcomas induced in C3H/HeJ mice with 3-methylcholanthrene, were employed to compare the relative immunogenic activity of intact tumor cells, 2.5% butanol extracts, and materials sequentially purified by preparative isoelectric focusing (pIEF), preparative isotachophoresis (pITP), and high performance gel permeation chromatography (HPGPC). Immunoprotective TSTA activity purified 50,000-fold by this protocol extended the effective dose range by four to five logs: 15 pg to 1.5 micrograms MCA-F or 1 pg to 10 ng MCA-D antigen-induced specific host resistance. However, despite the appreciable purification of TSTA, immunization with extracted materials only delayed neoplastic outgrowth. They induced neither immediate rejection nor only temporary progression of transplanted tumor cells. Thus, purified TSTA preparations by themselves lack the immunogenic properties of intact cells that result in maximal induction of tumor resistance.

“…To the best of our knowledge, no information is available about the PHA-induced expression of HLA Class II antigens and the functional activity in autologous MLR of T cells isolated from draining lymph nodes (LN-T) from patients with solid tumors. In view of the release of immunoregulatory factors from human tumors [10,14,19,20,26], in this study we have compared the induction of HLA Class II antigens and the functional properties in autologous and allogeneic MLR of T cells isolated from mediastinal lymph nodes and from peripheral blood of patients with lung carcinoma.…”

Section: Introductionmentioning

confidence: 99%

Abnormalities of T cells isolated from mediastinal lymph nodes and peripheral blood of patients with lung carcinoma: deficit in PHA-induced expression of HLA class II antigens and in autologous mixed lymphocyte reactions

Indiveri¹,

Pierri

Rogna

et al. 1986

A reduced percentage of T cells isolated from mediastinal lymph nodes and peripheral blood of patients with lung carcinoma acquired HLA Class II antigens following in vitro stimulation with PHA. Furthermore T cells were functionally abnormal in autologous and allogeneic mixed lymphocyte reactions (MLR). The immunoregulatory properties of HLA Class II antigens and autologous MLRs suggest that these abnormalities may affect the interaction of the host's immune system with tumor cells.