concanavalin-a and perfosfamide

We investigated the immunomodulatory effect of cyclophosphamide on the induction of suppressor cell activities in peripheral blood lymphocytes. The sera from advanced breast cancer patients as well as concanavalin-A (Con-A) induced suppressor cell activities in lymphocytes from healthy volunteers. Pretreatment of these lymphocytes, including CD4+ T cells with 4-hydroperoxycyclophosphamide (4-HC), an active form of cyclophosphamide, abrogated the induction of suppressor cell activities by either cancer sera or Con-A. A decrease in CD4+CD45RA+ suppressor/inducer T cells and reduction of Con-A induced suppressor cell activities were observed in breast cancer patients with metastases that were treated with cyclophosphamide (CPM). These results suggest that cyclophosphamide may modulate the immune responses of breast cancer patients by interference with suppressor/inducer T cells.

Topics: Breast Neoplasms; Concanavalin A; Cyclophosphamide; Female; Humans; T-Lymphocytes, Regulatory

The aim of the present study was to analyze further the immunopotentiating effects of low doses of oxazaphosphorines. We examined 4-hydroperoxycyclophosphamide (4-HC) and mafosfamide, which degrade spontaneously in water without requiring liver enzymes to become active. Both drugs, at concentrations ranging from 0.01 microM to 1 microM, enhanced mitogenic responses of human lymphocytes. Higher concentrations were toxic. Acrolein, which is one of the degradation products of oxazaphosphorines, had similar effects. Immunopotentiation was not monocyte-dependent. Attempts to inactivate released acrolein with human serum reduced toxicity but the immunostimulating property of the drugs remained Similar effects were noted when lymphocytes were exposed to acrolein dissolved in serum. 2-Mercaptoethane-sulfonate (mesna), which is highly reactive with acrolein, reduced the toxicity of solutions of both oxazaphosphorines and acrolein. Immunopotentiation was not clearly demonstrable since mesna itself enhanced the responses. Pretreatment of lymphocytes with 4-HC or mafosfamide did not reduce the capacity of concanavalin A to induce suppressor cells. It is speculated that acrolein may play a role in oxazaphosphorine-induced enhancements of immune responses.

Topics: Acrolein; Aldehydes; Antineoplastic Agents; Concanavalin A; Cyclophosphamide; Humans; Immunity, Cellular; Lymphocytes; Mitogens; Mitosis; T-Lymphocytes; T-Lymphocytes, Regulatory

Treatment of T cells in vitro with low concentrations of 4-hydroperoxycyclophosphamide (4-HC) is known to result in immunopotentiation of both T and B cell effector function in a manner analogous to that of cyclophosphamide administered in vivo. A previous study demonstrated that augmentation of polyclonal immunoglobulin secretion occurs following pretreatment of autologous collaborating T cells with low concentrations of 4-HC as a result of blockade of suppressor effector induction from suppressor precursor, both of which share the identical T4+,8-phenotype. The present study was undertaken to examine the effects of 4-HC on regulatory T-T interactions in mixed lymphocyte culture (MLC) responses and for allospecific cytotoxic T lymphocyte (CTL) responses. Induction of CTL and MLC proliferation were found to be sensitive to as little as 40 microM 4-HC, whereas CTL effector function was resistant to greater than or equal to 80 microM. CTL effectors were restricted to the T4-,8+ subset and the cells showing sensitivity to low and intermediate 4-HC concentrations were found to be T4+,8-. Secondary MLC and CTL responses displayed a similar 4-HC concentration-dependent inhibition following drug treatment of the T4+ T subset which could only be detected at suboptimal responder to stimulator ratios. This suggests that the mechanisms of CTL induction by a T4+ inducer cell in primary and secondary MLC responses and the sensitivity of induction of 4-HC are qualitatively similar. Pretreatment of T cells with less than or equal to 20 microM 4-HC for one hour prior to Con A activation totally blocked suppressor effector induction both for MLC and CTL function. In contrast, treatment with 80 microM 4-HC following Con A induction was without effect on differentiated T suppressor effector activity. Studies utilizing monoclonal antibody/complement depletion and panning techniques demonstrated that the suppressor precursor and differentiated suppressors for T effector function were restricted to the T4+,8-subset. These results support the hypothesis that regulatory inducer T cell function is significantly more sensitive to the inhibitory effects of low to intermediate concentrations of 4-HC than either the suppressor-cytotoxic precursors themselves or suppressor/cytotoxic effectors. Con A inducible suppressor cell precursor induction (mediated by the T4+,8-subset) demonstrated the greatest sensitivity to 4-HC (less than or equal to 20 microM) followed by inducers of primary

Topics: Cells, Cultured; Chromium Radioisotopes; Concanavalin A; Cyclophosphamide; Humans; Immunity, Cellular; Lymphocyte Culture Test, Mixed; T-Lymphocytes; T-Lymphocytes, Regulatory

The present study examined the effect of pulse treatment with the in vitro active synthetic derivative of cyclophosphamide (CY), 4-hydroperoxycyclophosphamide (4-HPCY), and exposure to X-irradiation on the in vitro Concanavalin A (ConA), lipopolysaccharide (LPS), and antigen-specific blastogenic responses of in vivo-primed lymph node cells. Primed lymph node cells from CY-pretreated, aggregated (A) human IgG-complete Freund's adjuvant (AHGG-CFA)-immunized mice were untreated, exposed to various doses of irradiation, or pulse treated with different concentrations of 4-HPCY before being cultured in medium alone or in medium containing HGG, ConA, or LPS. The results show that HGG-responding and LPS-responding cells exhibited similar dose-inactivation profiles following exposure to irradiation or pulse treatment with 4-HPCY. More than 75% of reactivity was eliminated by exposure to 100 rads or pulse treatment with 20 microM 4-HPCY. In contrast to preculture pulse treatment with 4-HPCY, however, when primed lymph node cells were cultured in medium containing 4-HPCY (culture treatment) LPS-responding cells were shown to be more sensitive to inactivation than HGG-responding cells. The data further show that the effect of low-dose irradiation and of culture treatment with 4-HPCY on the HGG-specific response of primed lymph node cells was additive, suggesting that these agents inactivate different cell subtypes that contribute to the HGG-specific response in vitro.

Topics: Animals; Antigens; Concanavalin A; Cyclophosphamide; Dose-Response Relationship, Immunologic; Dose-Response Relationship, Radiation; Epitopes; Female; Freund's Adjuvant; Humans; Immunoglobulin G; Kinetics; Lymph Nodes; Lymphocyte Activation; Lymphocytes; Mice; Mice, Inbred C57BL

Treatment in vitro of human peripheral blood lymphocytes (PBL) with ConA induced the generation of suppressor cells which inhibited T cell blastogenic response to ConA and of allogeneic response in the mixed lymphocyte reaction (MLR). Treatment of PBL with 4-hydroperoxycyclophosphamide (4-HPCy) before incubation with ConA markedly decreased the generation of suppressor cells by ConA. The effect of 4-HPCy on generation of suppressor cells was more pronounced in the test of ConA stimulation than in the MLR. Treatment with 4-HPCy had no effect on suppressor cells already induced as shown by incubation of PBL with 4-HPCy after incubation with ConA.

Topics: Cells, Cultured; Concanavalin A; Cyclophosphamide; DNA Replication; Humans; Kinetics; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; T-Lymphocytes, Regulatory

The alkylating agent cyclophosphamide may suppress or enhance immune responses in vivo but is inactive in vitro unless metabolized by microsomal enzyme activation. 4-hydroperoxycyclophosphamide (4-HC) is a synthetic compound that is spontaneously converted in aqueous solution to the active metabolites. In this report, we examined the in vitro sensitivity of functional human T cell subsets to 4-HC in a polyclonal B cell differentiation assay and in the generation of mitogen-induced suppressor cells for effector B cell function. Con A-induced T suppression of B cell differentiation is completely abrogated by a 1-h pretreatment of T cells at very low concentrations of between 10(-2) and 20 nmol/ml, whereas inducer T cell function is sensitive only to concentrations in greater than 40 nmol/ml. The effects of 4-HC on suppressor T cells appear to occur at concentrations that do not result in DNA cross-linking or decreased blastogenesis. Con A-induced T suppressors are generated from within the OKT4+, OKT8- subset and are sensitive to low-dose 4-HC only before activation, whereas differentiated suppressor cells are resistant to concentrations in greater than 80 nmol/ml. Low-dose 4-HC pretreatment of the B cell population results in abrogation of immunoglobulin secretion when treated B cells are cocultured with unfractionated T cells, however, this effect is completely reversible if pretreated B cells are cocultured with T cells devoid of suppressor activity. These results demonstrate that human presuppressor cells for B-effector function differentiate in response to Con A from the OKT4+, OKT8- subset and are exquisitely sensitive to low concentrations of CYP whereas mature suppressor and inducer functions are resistant to all but very high concentrations in vitro. The differential sensitivity of functional T and B cell subsets to 4-HC in vitro can be a very useful probe in dissecting immunoregulatory interactions with man.

Topics: B-Lymphocytes; Cell Separation; Concanavalin A; Cyclophosphamide; Humans; Immunoglobulins; Lymphocyte Activation; Lymphocyte Cooperation; T-Lymphocytes