Benny Leshem

Functional clonal deletion versus active suppression in transplantation tolerance induced by total-lymphoid irradiation

Transplantation tolerance and stable chimerism were established in adult mice conditioned with a short course of total-lymphoid irradiation (TLI) followed by infusion of 30 X 10(6) allogeneic bone marrow cells. Spleen cells of tolerant mice could not exert a proliferative or cytotoxic response against host-type cells in vitro and were unable to induce graft-versus-host reaction in secondary host-type recipients. The degree of suppression assessed by coculturing tolerant splenocytes in vitro in the one-way mixed lymphocyte reaction was quite variable--and, in some cases, was not at all demonstrable, although tolerance was clearly maintained. Suppression, when apparent, could not be ascribed to T lymphocytes. Suppressor cells were found to bind soybean agglutinin and could be separated from the nonsuppressive cells by means of this lectin. Dissociation of the suppressive population (SBA+ cells) from that which is normally alloreactive (SBA- cells) resulted in a suppressor cell-depleted fraction that was still unable to respond to host-type cells but regained reactivity to unrelated cells. Limiting dilution analysis of chimeric splenocytes revealed markedly reduced frequencies of cytotoxic T lymphocyte precursors (CTL-P) directed against host-type cells, as compared with normal splenocytes reacting against the same target cells. This difference was accentuated when these cells were sensitized to host-type target cells prior to plating in limiting dilution cultures. In 1:1 mixing experiments of normal and chimeric splenocytes, there was no evidence of any in vitro suppressive activity to account for hyporeactivity of chimeric cells against host-type cells. Thus, maintenance of TLI-induced tolerance seemed not to be mediated primarily through an active suppressor cell mechanism.

Prediction by a modified mixed leukocyte reaction assay of graft-versus-host disease and graft rejection after allogeneic bone marrow transplantation

In this report we describe a modified, sensitive MLR test that appears to detect fine antigenic disparities between HLA-identical siblings confirmed as such by serology and the standard MLR test. In a group of 40 consecutive allogeneic bone marrow transplants, reactivity detected by the modified MLR test correlated with the development of rejection of matched marrow grafts and onset of acute graft vs. host disease (aGVHD). Thus, 13/15 positively reacting patient/donor pairs developed one of these complications (P < 0.001), while only 2/25 developed aGVHD in the negatively reacting group. This test may be useful for selecting the most compatible donor when several potential donors are available.

Chemo-immunotherapy of murine tumors using interleukin-2 (IL-2) and cyclophosphamide

SummaryThe antitumor effect of interleukin-2 (IL-2), alone and in combination with cyclophosphamide was assessed in mice with established sarcoma (MCA 105, H-2b), carcinoma (M109, H-2d) and T lymphoma (PIR-2, H-2b). Whereas administration of IL-2 alone (5×104–10×104 U, i.p. twice daily, for 4–8 consecutive days) prolonged the survival of mice with the solid neoplasms, it enhanced tumor growth and decreased survival of mice with the lymphoma. In the PIR-2 lymphoma, no IL-2 receptor (TAC) could be detected, nor could we demonstrate IL-2 tumor growth stimulation in vitro. A synergistic therapeutic effect was achieved in mice with the solid tumors, but not in mice with the lymphoma, only when IL-2 was given 1–4 days after cyclophosphamide (100–200 mg/kg). Conversely, administration of IL-2 1–4 days prior to cyclophosphamide resulted, in all three tumor systems, in enhanced tumor growth and in decreased survival as compared with mice receiving cyclophosphamide alone. Similarly, treatment with IL-2 both before and after cyclophosphamide was less efficacious than a single course of IL-2 given after-wards. It is concluded that for maximal therapeutic efficacy, IL-2 should be administered following chemotherapy, and that certain tumors may respond adversely to IL-2 treatment.

In vitro elicitation of cytotoxic response against a nonimmunogenic murine tumor by allosensitization

SummaryThe murine lymphoma (thymoma) PIR-2 of C57BL/6 origin, primarily induced in our laboratory by fractionated X-ray irradiation, has been shown to be nonimmunogenic by its failure to immunize syngeneic mice in vivo or to evoke a cytotoxic response in primary mixed lymphocyte-tumor cell cultures (MLTC) in vitro. We were able, however, to demonstrate the existence of anti-PIR-2 cytotoxic cells among allogeneic-primed C57BL/6 responding lymphocytes using the technique of limiting dilution cultures (LDC). The frequency of anti-PIR-2 cytotoxic cells among C57BL/6 lymphocytes sensitized against BALB/c splenocytes in mixed leukocyte culture (MLC) was 1/20 to 1/40, and the cytotoxic activity of positive LDC wells against PIR-2 reached 60% as determined by a 4-h 51Cr-release assay. The frequency of anti-PIR-2 cytotoxic cells could be increased two- to 10-fold (up to 1/4) by removing nylon-wool-adherent cells from the primed cell population and/or by enriching the primed lymphoblast population on a Percoll density gradient. Anti-PIR-2 cytotoxic cells were found to be Thy1+; Lytl−2+ cells. Clones isolated from the LDC wells manifested strong cytotoxic activity toward PIR-2 cells and the stimulating BALB/c splenocytes but not against other H-2b tumor lines or C57BL/6 splenocytes. We suggest that the procedure of allostimulation in MLC-LDC is an effective in vitro means of generating highly reactive cytotoxic cells against poorly immunogenic neoplasms.

Chemo-Immunotherapy of murine solid tumors: Enhanced therapeutic effects by interleukin-2 combined with interferon α and the role of specific T cells

SummaryThe aim of the present study has been to assess the therapeutic efficacy of various cytokines, singly or in combination, with and without chemotherapy (cyclophosphamide, Cy), in mice carrying advanced, weakly immunogenic tumors (MCA-105 sarcoma, M109 carcinoma). Treatment of animals with i.p. growths or experimental pulmonary metastases began 8–18 days after i.p. or i.v. tumor cell inoculation respectively. None of the cytokines tested [interleukin-2 (IL-2), interferon α (IFNα), tumor necrosis factor α (TNFα) and macrophage-colony-stimulating factor (M-CSF)] nor Cy had by itself a significant curative effect. A synergistic therapeutic effect was obtained with IL-2 or IFNα (but not with TNFα or M-CSF) in combination with Cy. The most efficacious regimen (65%–90% cure of mice carrying i.p. tumors) was the combination of Cy+IL-2+IFNα. Preliminary experiments suggested that sequential administration of these cytokines might be more beneficial than concurrent administration. Following successful immunotherapy, long-term (3–6 months) survivors showed a tumor-specific resistance to a second tumor challenge and their spleen contained an increased number of specific antitumor cytotoxic T lymphocyte precursors (5- to 20-fold, compared to control mice). In vitro and in vivo cell-depletion experiments using monoclonal antibodies revealed that T cells (primarily CD8), but not NK cells, are crucial for the therapeutic effects. This study indicates that a potent specific antitumor T cell immunity can be elicited against advanced weakly immunogenic tumors by combining chemotherapy (Cy) with IL-2 and IFNα.

A short human and mouse MLR assay utilizing lymphokine (IL-2, IL-3) secretion as an early activation event

The mixed leukocyte reaction is the only functional in vitro assay currently employed for confirmation of MHC matching between bone marrow recipients and their prospective donors and for MHC class II (HLA-Dw) typing. This assay is, however, time-consuming (6 days for human MLR), whereas for clinical purposes results are often required much earlier. In an attempt to shorten the MLR incubation period, we tested IL-2 (in human MLR) and IL-2/IL-3 (in mouse MLR) production as an indication of early stages of T cell activation. We here describe a shorter assay in which IL-2 and IL-3 secretion during MLR was assessed by adding the respective lymphokine-dependent cell lines either to the MLR supernatants or directly to the original MLR cultures, using the colorimetric (3-[4,5 Dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) (MTT) technique or the 3H-thymidine incorporation assay. In both human and mouse MLR systems, lymphokine production peaked at 24–48 hr after culture initiation, allowing tests to be completed within 48 to 72 hr. Weak MLR responses, as detected by lymphokine production, could be considerably amplified by irradiating (250–1000 cGy) the responder cells and by adding heparin (1–10 U/ml) to the cultures. The results obtained by this novel procedure correlated with those obtained by the standard 6-day human MLR assay in over 250 combinations tested thus far, and therefore it may replace the standard MLR procedure.

BONE MARROW TRANSPLANTATION WITH T-CELL-DEPLETED GRAFTS

The potential role of donors mature T lymphocytes on the recovery of various immunological functions and hematopoiesis was investigated in lethally irradiated BALB/c mice by studying reconstitution with normal, as compared with T-cell-depleted, syngeneic marrow grafts. Recovery of total, as well as mononuclear, peripheral white blood cell counts, platelets, hemoglobin levels, proportion of Thy 1.2+ cells, responses to concanavalin A, phytohemagglutinin and lipopolysaccharide, mixed lymphocyte response, cell-mediated lympholysis response, anti-SRBC agglutinins and natural killer activity were basically similar in recipients of unmanipulated (as compared with T cell depleted) syngeneic marrow grafts. The data suggest that in a syngeneic murine bone marrow transplantation setting, mature donor T lymphocytes do not seem to play a major role in immunohemopoiesis. Normal T cell number and T-cell-dependent immune function can be readily regenerated out of the stem cell reservoir of adult donors following transplantation into lethally ablated recipients.

IRON CHELATORS: CORRELATION BETWEEN EFFECTS ON PLASMODIUM SPP. AND IMMUNE FUNCTIONS

Iron chelating agents, which permeate through erythrocytic and parasite membranes, are effective against Plasmodium falciparum in vitro. However, the protective effect in humans is transient. We examined the antiplasmodial capacity of several iron chelators in vitro and in vivo. The chelators 3/3hb/2m and 3/2hb/b (together, MoB) were more effective against P. falciparum in vitro than desferrioxamine (DFO) and Salicylaldehyde isonicotinoyl hydrazone (SIH) (together, DoS). Despite similar pharmacokinetics of all iron chelators, mice infected with Plasmodium vinckei and treated with MoB succumbed to malaria, whereas DoS-treated mice survived. However, even in the surviving mice, peak parasitemias were above 30%. These results indicate that the direct effects of the drugs on the parasites were not responsible alone for the complete recovery of the mice. We suggest that the recovery is related to differential effects of the drugs on various immune functions. We concentrated on the effect of the iron chelators on B cell and T cell proliferation and on allogeneic stimulation (MLR), interleukin-10 (IL-10), γ-interferon (γ-IFN), tumor necrosis factor-α (TNF-α), and radical production. All the iron chelators examined inhibited the in vitro proliferation of B cells and T cells, and MLR. This may explain why iron chelators are only slightly efficient in treating human malaria. However, the inhibitory effects of MoB on B cell and T cell proliferation and on MLR were more pronounced than those of DoS. In addition, the release of free radicals by effector cells was inhibited to a greater extent by MoB than by DoS. These results may explain why MoB, which was more efficient in vitro, was not effective in vivo. The DoS effects on the in vitro secretion of cytokines correlate with their in vivo effect; there was a decrease of IL-10 and a parallel increase in γ-IFN and TNF-α production by human mononuclear cells. MoB, which could not rescue the animals from malaria, did not affect IL-10 and TNF-α, but reduced γ-IFN levels. Identical results were obtained when using monocytes instead of mononuclear cells (except for γ-IFN, which is not produced by monocytes). Our results indicate that an iron chelator, or any antiparasitic drug that kills the parasites in vitro, should also be selected for further evaluation on the basis of its reaction with immune components; it should not interfere with crucial protective immunological processes, but it may still alleviate parasitemia by positive immune modulation.

Heat inactivation of fetal calf serum is not required for in vitro measurement of lymphocyte functions.

This study was undertaken to test whether fetal calf serum (FCS) must be heat inactivated before use in tissue culture. We tested various immune functions of lymphocytes growing in medium containing non-treated and heat-inactivated FCS. The data clearly show that heat inactivation of the serum is not mandatory. In some cases, the addition of untreated FCS resulted in elevated response levels, while maintaining immune function specificity.

Iron chelators as drugs against malaria pose a potential risk.

Iron is involved in different processes which seem to be unrelated to each other: metabolic pathways, cell cycle, radical production, and apoptosis. However, all these processes are interrelated by flow of signals in cells and communication among cells and organs. Consequently, an intervention with an iron chelator has direct and indirect effects on various cell functions and should be carefully examined before being applied. In plasmodial infections, there are additional complications which make it even more difficult to discriminate individual effects of iron chelation. The approach of developing an iron chelator for treatment of malaria, based on a temporary effect on parasitemia, is naive.