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D Collavo

Publications and source records attributed to D Collavo.

At least 19 recordsLinked to original sources
PubMed1992

Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases.

Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricin-treated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.

Animals↗
PubMed1992

Role of adhesion molecules in the immune reaction to M-MSV-induced tumors.

We have investigated the in vivo role of 2 different adhesion molecules, LFA-1 and LECAM-1, in the immune reaction to Moloney-murine-sarcoma-virus(M-MSV)-induced tumors, which undergo a peculiar spontaneous regression due to generation of a strong virus-specific cytotoxic-T-lymphocyte(CTL) response. Repeated administration of anti-LFA-1 monoclonal antibody (FD441.8 MAb), i.p. or at the site of virus inoculation, enhanced tumor growth and delayed regression, while i.p. administration of anti-LECAM-1 MEL-14 MAb gave rise to tumors that grew progressively and caused host death. Evaluation of the immunological response in MAb-treated mice showed reduced generation of virus-specific CTL precursors (p) in the spleen of animals given FD441.8 MAb i.p.; CTLp frequency in locally treated mice overlapped with that of control mice injected with virus only. FD441.8 MAb treatment did not interfere with CTL homing in the tumor, since the frequency of M-MSV-specific CTLps in sarcomas was similar in treated and control mice. Cytofluorimetric analysis indicated that the majority of tumor-infiltrating lymphocytes (TIL) from MAb-treated mice were covered by anti-LFA-1 MAb, and lacked cytotoxic activity when assayed against target cells bearing relevant tumor antigens. Instead, in mice injected i.p. with MEL-14 MAb, a very low frequency of CTLps was detected in lymph nodes draining the tumor area, and within the tumor. Our results indicate that enhanced tumor growth, depending on the MAb used, is the resultant of an inhibitory effect on different T-lymphocyte functions. Tumor progression in anti-LFA-1 MAb-injected mice is explained mostly by blockage of CTL lytic activity at the tumor site; in mice receiving i.p. MEL-14 MAb treatment, by the failure of naive T lymphocytes to enter peripheral lymph nodes and subsequently by the lack of generation of tumor-specific CTLs.

Animals↗
PubMed1992

The in vivo role of leukocyte function-associated antigen-1 in cytotoxic cell activity against tumors induced by Moloney-murine sarcoma/leukemia retroviral complex.

The in vivo role of LFA-1 molecule in the immune reaction to a murine retrovirus-induced tumor was investigated. Local and systemic treatment with high doses of anti-LFA-1 monoclonal antibody led to tumor progression by blocking CTL interaction with tumor cells without interfering with CTL generation and localization in the tumor mass.

Animals↗
PubMed1991

Moloney murine leukemia virus tolerance in anti-CD4 monoclonal antibody-treated adult mice.

Adult mice injected with Moloney murine leukemia virus (M-MuLV) 1 day after a short term treatment with anti-CD4 mAb developed T cell lymphomas, or sarcomas when rechallenged with Moloney murine sarcoma virus (M-MSV). Neoplastic development was correlated with virus spread, as thymic and peripheral T and B lymphocytes promptly expressed M-MuLV-induced Ag after virus introduction; no virus-specific CTL generation was detected in MLTC. This failure, which was selective for M-MuLV-induced Ag, persisted throughout the life span of the mice, and was not sustained by suppressor cell activity. The frequencies of splenic virus-specific CTL precursor varied in relation to time after virus injection; in the first postinjection month, frequencies were similar to those in nonimmune control mice, while at 4 mo post-virus exposure, they showed a striking reduction. These findings indicate that a transient functional impairment of CD4+ cells at the time of retrovirus injection provided the appropriate milieu for tolerance induction in both the peripheral mature and intrathymic T cell compartments.

Animals↗
PubMed1991

An improved method for the detection of DNA fragmentation.

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

Animals↗
PubMed1991

Adoptive immunotherapy of experimental tumors using cytotoxic lymphocytes to carry and deliver toxins.

Adoptive transfer of specifically sensitized lymphoid cells constitutes a potential tool for specific cancer immunotherapy, however, the requirement of syngeneic or autologous specifically reactive cells has limited its use in the treatment of human cancer. In several mouse tumor models, large amounts of lymphokine activated killer (LAK) cells associated with high doses of interleukin 2 (IL-2) are able to mediate the regression of established pulmonary and hepatic metastases. Since LAK cells are more easily generated than specifically sensitized lymphocytes and show broad tumor specificity, this approach has been applied in humans to treat cancer, but the acute toxicity associated with the high doses of IL-2 administered represents an important drawback to its clinical use. The observation that lymphocytes can internalize ricin, a toxic plant protein, and then release it in an active form, capable of destroying other cells, led us to investigate the possibility of using antigen-specific cytotoxic T lymphocytes (CTL) or LAK cells as carriers of toxic substances to the tumor site. In our experimental models we observed that tumor-specific CTL or LAK cells can be used to deliver ricin into the tumor mass and cause temporary tumor growth inhibition.

Animals↗
PubMed1990

Induction of Moloney murine sarcoma virus tolerance in adult mice by anti-CD4 monoclonal antibody treatment.

The role of CD4+ cells in the immune response to Moloney murine sarcoma/leukemia virus complex was studied in adult mice undergoing long-term CD4+ cell functional depletion by treatment with anti-CD4 H129.19 monoclonal antibody (immunoglobulin G2a, kappa). Adult mice given injections of Moloney murine sarcoma/leukemia virus complex 1 day or 2 wk after monoclonal antibody treatment died with progressing sarcomas at the inoculation site; mice challenged 4 wk after such treatment, when CD4+ cells recovered their functional activity, behaved like conventional mice; i.e., they spontaneously regressed the virus-induced sarcomas. Mice with progressing tumors did not generate virus-specific cytotoxic T-lymphocytes, despite a normal cytotoxic T-lymphocyte response to unrelated antigens, and they became virus carriers as demonstrated by Moloney murine leukemia virus antigen expression on their lymphoid cells a few days after virus injection. Moreover, the observed T-lymphocyte unresponsiveness was not due to the activity of specific suppressor T-cells. The findings indicate that the transient functional depletion of CD4+ cells at the time of virus administration provides appropriate environmental conditions for the spread of virus and facilitates tolerance induction.

Animals↗
PubMed1990

Extracellular ATP as a possible mediator of cell-mediated cytotoxicity.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse target cells by a specialized mechanism that involves the release of soluble 'killer' mediators. While several possible mediators have been demonstrated, it is likely that others have yet to be identified. For more than two decades it has been known that exogenous ATP is lytic to many cell types. In this article Francesco Di Virgilio and colleagues propose that extracellular ATP may be a mediator of cytotoxic cell-dependent lysis.

Adenosine Triphosphate↗
PubMed1989

Responses of mouse lymphocytes to extracellular adenosine 5'-triphosphate (ATP). Lymphocytes with cytotoxic activity are resistant to the permeabilizing effects of ATP.

The effects of extracellular ATP on plasma membrane permeability in mouse lymphocytes were studied with plasma membrane depolarization, uptake of ethidium bromide, and release of lactate dehydrogenase as indicators of increased permeability. Extracellular ATP induced sustained depolarization of plasma membrane potential as well as uptake of low m.w. fluorescent markers in mouse lymphocytes derived from thymus and spleen, and in two lymphoma lines YAC-1 and MBL-2. The fully ionized form ATP4- rather than MgATP2- mediated the increased permeability of the plasma membrane. Although prolonged exposure to exogenous ATP ultimately lysed the lymphocytes, two CTL populations (CHM-14 clone and CTLL-2 line) and IL-2-treated spleen lymphocytes with unrestricted killing activity were highly resistant to the permeabilizing action of extracellular ATP at all concentrations tested. In addition, CTL derived from primary immune peritoneal exudate and enriched by in vitro culture for 5 days in the presence of specific stimulator cells were also resistant to this permeabilizing effect. These findings show that exogenous ATP has a lytic effect on mouse lymphocytes but not on CTL, and suggest a role for ATP in cell-mediated cytotoxicity.

Adenosine Triphosphate↗
PubMed1989

Interaction of lymphokine-activated killer cells with susceptible targets does not induce second messenger generation and cytolytic granule exocytosis.

CTL activation by specific targets leads to a rapid rise of inositol phosphates (InsPs) and of cytoplasmic-free Ca2+ concentration ([Ca2+]i). While these events are considered necessary to trigger granule secretion, Ca2+-independent cytolytic mechanisms have been recently proposed in addition or as an alternative to the classical Ca2+-dependent exocytosis model. We observed that lymphokine-activated killer (LAK) cells, obtained after stimulation with supraoptimal concentrations of IL-2 in short- or long-term cultures, kill susceptible targets in the absence of a [Ca2+]i rise and InsP3 formation. Moreover, LAK cell-mediated lysis was not associated with an increase in cytotoxic granule exocytosis, as evaluated by BLT-esterase release into the culture supernatant. Furthermore, using an antigen-specific CTL clone, which acquires LAK-like activity when cultured in medium containing high IL-2 doses, second messenger generation and cytolytic granule content secretion were not detected during lysis of unrelated target cells, while killing of specific targets triggered both these processes. These findings suggest that two lytic pathways may coexist in the same effector cells: a second messenger-dependent pathway involving degranulation, which is activated after TCR interaction with specific targets, and another pathway, independent of any known second messenger generation, responsible for unrelated target cell lysis.

Animals↗
PubMed1989

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity.

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.

Animals↗
PubMed1989

In vitro induction of immunological tolerance.

IL-2 was previously shown to induce cytotoxic effectors with a broad spectrum of target specificities in thymus and spleen cell cultures. This study was designed to show whether T cells activated by H-2 allogeneic cells in MLC or by syngeneic tumor cells in MLTC are also potential targets for these cytotoxic effectors. We found that thymocytes activated in vitro for 5 days by rIL-2 were capable of killing tumor cells as well as activated T cells. Thymocytes activated by IL-2 were accordingly utilized as a means of effecting clonal deletion of T cells activated by H-2 allogeneic target cells in MLC. To establish whether the unresponsiveness is specific. IL-2-activated thymocytes were added as third party cells to MLC and MLTC. The results showed that both T cells, proliferating in response to H-2 allogeneic cells, and CTL, reactive against syngeneic tumors or H-2 allogeneic cells, are eliminated from the T cell pool. Only alloreactive T cells are specifically eliminated in MLC by IL-2-activated thymocytes, as the remaining T cells are capable of proliferating and generating CTL in response to antigenically unrelated third party allogeneic cells. The possibility that unresponsiveness might be due to soluble factors was ruled out by studies performed with a diffusable "chamber insert" culture system. The results provide evidence that IL-2-activated thymocytes induce in vitro T cell tolerance.

Animals↗
PubMed1988

Monoclonal antibody against IFN-gamma inhibits Moloney murine sarcoma virus-specific cytotoxic T lymphocyte differentiation.

The role of autochthonous IFN- production was evaluated in immune reactions to Moloney murine sarcoma virus (M-MSV)-induced tumors which are characterized by spontaneous regression mainly caused by virus-specific CTL activity. A functional IFN- depletion, induced by repeated administration of mAb anti-IFN- at the site of virus inoculation, prevented tumor regression in M-MSV-injected mice. Moreover, this antibody inhibited in vitro both proliferation and differentiation of M-MSV-specific T lymphocytes obtained in bulk cultures, but not growth and lytic activity of the already differentiated virus-specific CTL clone CHM-14 stimulated with rIL-2 and relevant tumor Ag. In addition, in mice receiving mAb treatment the frequency of M-MSV-specific CTL precursors, evaluated by means of limiting dilution analysis, was strongly reduced in comparison with that of control mice injected only with virus. Because CTL secrete IFN- following antigenic stimulation, the possibility that non-T effector cells recruited by this lymphokine might mediate tumor regression was also considered. Adoptive immunotherapy experiments, performed in T cell-deficient (Tx + BM) and in sublethally irradiated mice, demonstrated that transfer of CHM-14 CTL clone, which secretes IFN-, was able to counteract M-MSV tumor growth despite the local mAb anti-IFN- treatment which may have prevented host cell recruitment. Moreover, repeated local rIFN- inoculations in Tx + BM mice did not counteract M-MSV tumor progression, thus confirming that other IFN- properties such as non-T cell recruitment, antiviral or anti-proliferative IFN- activities have little or no relevance when M-MSV-specific CTL are lacking. On the whole, these results indicate that in M-MSV-injected mice, tumor enhancement after mAb anti-IFN- treatment is principally caused by impaired differentiation of virus-specific CTL precursors.

Animals↗
PubMed1988

HIV-mediated immunodepression: in vitro inhibition of T-lymphocyte proliferative response by ultraviolet-inactivated virus.

In order to assess whether the human retrovirus HIV, like other animal retroviruses, is endowed with intrinsic immunosuppressive activity, we studied the effects of noninfectious, uv-irradiated virus on in vitro lymphocyte function. uvHIV preparations inhibited T-cell proliferation to mitogens and alloantigens, as well as mitogen-driven IL-2 production. The inhibitory effect, which was not exerted by uv-irradiated HTLV-I, was apparently not due to a decrease in cell viability and was likely associated with thermoresistant viral component(s). The suppression proved to be selective for T-cell responses, while sparing other lymphocyte functions, such as the B-cell proliferative response to a selective B-cell mitogen. The inhibitory effect of uvHIV was not counteracted by a substantial reduction in the number of monocytes or by indomethacin. Moreover, IL-1 production by monocytes was not affected upon virus incubation. On the other hand, the proliferative response of both CD4+ and CD8+ T-cell clones was inhibited by uvHIV, suggesting that T cells represent the actual target for the inhibitory effect. Although a sizeable decrease in IL-2 production was observed following uvHIV incubation, exogenous IL-2 was not capable of reversing the virus-induced suppression of the proliferation. The possibility that the immunosuppressive activity of noninfectious HIV contributes to the T-cell defect in infected patients by mechanisms other than the cytopathic effect on CD4+ T lymphocytes is discussed.

Animals↗
PubMed1987

Calcium and inositolphosphates in the activation of T cell-mediated cytotoxicity.

Reports from a number of laboratories have shown that mAbs against the T3-Ti receptor complex cause an increase in cytosolic-free Ca2+ [( Ca2+]i) and the hydrolysis of phosphatidylinositolbisphosphate (PIP2) in CTLs. In the present report we show that activation of CTLs by their specific targets causes: (a) release of Ca2+ from intracellular stores; (b) transient formation of inositol trisphosphate (InsP3); and (c) an increased permeability to Ca2+ of CTL plasma membrane. Killing of unrelated targets could be induced by cocentrifugation of the unrelated targets with CTLs in the presence of A23187 or PMA. We conclude that: (a) activation of CTLs by specific antigens triggers the generation of the same intracellular mediators generated by stimulation of lymphocytes with anti-T3-Ti receptor antibodies and/or with polyclonal mitogens; and (b) intracellular signals that mediate the delivery of the lethal hit by CTLs are indistinguishable from those that induce cell proliferation.

Animals↗