ABSTRACT
A soluble factor which augments the expression of major histocompatibility complex class I (MHC I) antigens on a number of murine tumor cell lines, has been isolated from the culture supernatants of mixed lymphocyte reaction of spleen cells derived from C57B1/6, Balb/c and Swiss mice. The factor, termed MHC-augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography and reverse phase HPLC. MHC-AF activity is associated with an 18 kDa molecule. MHC-AF activity was resistant to pH 2.0 treatment and partially purified MHC-AF preparations did not have any activity in L929 cell/vesicular stomatitis virus (VSV) interferon bioassay system. Antibodies to IFN-gamma did not block the activity of MHC-AF. These results indicate that a MHC-AF distinct from IFN-gamma, is produced by mouse spleen cells undergoing a mixed lymphocyte reaction.
Subject(s)
Mice , Animals , Antibodies/pharmacology , Chymotrypsin/metabolism , Chymotrypsin/chemistry , Comparative Study , Concanavalin A/pharmacology , Hot Temperature , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/drug effects , Interferon-gamma/pharmacology , Interferon-gamma/metabolism , Interferon-gamma/immunology , Lymphocytes/physiology , Mice, Inbred BALB C , Mice, Inbred C57BL , Proteins/pharmacology , Proteins/metabolism , Proteins/isolation & purification , Spleen/cytology , Trypsin/metabolism , Trypsin/chemistry , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/drug effectsABSTRACT
Human peripheral blood mononuclear cells (PBMCs) activated with Con-A release a soluble factor which augments the expression of class I major histocompatibility complex (MHC) antigens by a variety of tumour cells. Previous attempts to purify this factor called MHC-activating factor (AF) (MHC-AF) made us realize that the presence of large numbers and quantities of irrelevant fetal calf serum proteins in the culture supernatants of the activated human PBMCs, interfered with the purification procedure. It was therefore necessary to standardize the use of a serum free culture medium to generate human MHC-AF. In the present communication we have tried several types of culture media and have identified DCCM-2 as the most suitable culture medium to generate human MHC-AF. MHC-AF generated in DCCM-2 medium appears to be a protein molecule resistant to pH 2 treatment but sensitive to heat treatment (56°C × 45 min) and treatment with proteolytic enzymes trypsin and chymotrypsin.
ABSTRACT
Culture supernatants of Concanavalin A activated human peripheral blood mononuclear cells were found to contain a factor which induced proliferative response in normal peripheral blood mononuclear cells. This proliferation-inducing factor specifically induced and sustained proliferation of purified human NK cells but not of T or B cells. Although interleukin 2 (IL12) also has proliferation-inducing effects on NK cells, the partially purified proliferationinducing factor preparations contained no measurable IL2 contamination. Moreover, neutralizing anti-IL2 antibodies did not block the growth effect of proliferation-inducing factor on purified human NK cells. Other cytokines which were tested, including IL4, IL6, IL7, IL12, TNF and IFN, were all found to be inactive in the proliferation-inducing factor assay. While proliferation-inducing factor by itself had no effect on T-cell proliferation, IL2-induced proliferation of T cells was significantly enhanced in the presence of proliferation-inducing factor, as was IL2-induced NK-cell proliferation. NK cells could be maintained in culture for at least a month in the presence of proliferation-inducing factor alone, but the cells lost their cytolytic activity after 3-4 weeks in culture. Addition of IL2, to NK cells which had been cultured in the presence of proliferation-inducing factor, restored their cytotoxicity. Proliferation-inducing factor activity was partially purified on an anion exchange HPLC column. The molecular weight of proliferation- inducing factor appeared to be about 10 kDa, based on its elution profile on a sizing HPLC column. Our results indicate that proliferation-inducing factor is a novel NK-cell proliferationinducing factor.
ABSTRACT
A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC-AF) has been partially purified by Sephadex G-100 column chromatography, preparative isoelectric focusing and HPLC with ion-exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6·0. Interferon (IFN)-α, ß, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC-AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-ß and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2·0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.