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The mechanism of unresponsiveness in lymphocytes specifically selected by antigen in normal rats.

The immunological potentialities of populations of thoracic duct lymphocytes collected from rats recently challenged with sheep erythrocytes were compared with those of lymphocytes from rats immunologically tolerant of this antigen. Both "selected" and tolerant populations fail to mount immune responses to sheep erythrocytes in irradiated syngeneic hosts but can do so if transferred to semi-allogeneic recipients. If co-cultivated with normal lymphocytes in irradiated hosts, both types of population can modulate the responsiveness of the former to sheep erythrocytes. Supplementation of normal lymphocytes with "selected" populations collected earlier than 28 hours after challenge of the donors tends to result in augmented responses while later collections are more likely to be associated with suppression. Consequently, it is suggested that "selected" populations are unresponsive because of the presence of specifically activated cells rather than as a result of their depletion. The influence of "selected" populations on the responsiveness of normal lymphocytes is attributed to this content of activated cells.

Animals↗

Facilitation of the growth of an allogeneic tumour by suppressor cells in newborn rats.

The intravenous injection of as few as 15 Walker tumour cells into newborn rats consistently resulted in the development of pulmonary metastases and the death of the recipient within 2 weeks. Neither the outcome of tumour cell injection nor the interval until death could be modified by transferring 2 x 10(7) lymphocytes from tumour-immune adult rats to the neonataal hosts. In contrast with this failure to transfer adoptive anti-tumour immune responses to intact recipients, the administration of 350 rad irradiation before transfer of 10(6) immune lymphocytes constantly afforded protection against inoculated tumour cells. The simultaneous transfer of neonatal thymus cells with immune lymphocytes interfered with the establishment of an adoptive response in the irradiated newborn. Intiation of a graft-versus-host response in F1 hybrid neonates by injecting parental strain lymphocytes conferred resistance to tumour growth of the recpient, the magnitude of this effect increasing with the strength of the graft-versus-host reaction.

Animals↗

Chemotherapy does not nullify the ability of donor lymphocyte infusions to mediate graft-versus-host reactions.

Two patients with multiple myeloma in relapse after allogeneic BMT received donor lymphocyte infusions (DLI) but later required chemotherapy for treatment of myeloma-related complications. In both patients, recovery from chemotherapy-induced aplasia was accompanied by manifestations of graft-versus-host reactions. The first patient developed grade II acute GVHD and a complete remission which has lasted >22 months. The second patient developed grade III acute GVHD but died with co-existing GVHD and extensive extramedullary myeloma. These results demonstrate that chemotherapy does not nullify the ability of donor lymphocytes to mediate graft-versus-host reactions.

Adult↗

Maintenance of immune memory to the hepatitis B envelope protein following adoptive transfer of immunity in bone marrow transplant recipients.

Adoptive transfer of immunity against hepatitis B surface antigen (HBsAg) has been documented in mice and humans. In the present study, we report long-term follow-up of antibodies to HBsAg in humans who received allogeneic bone marrow transplantation (BMT) from donors immunized with HBsAg. BM donors were immunized with recombinant HBsAg. BM or PB cells were transplanted to HLA matched recipients. Recipients were followed for anti-HBs seroconversion. Control groups included non-immunized or rHBsAg immunized healthy adults as well as individuals that had had hepatitis B and recovered spontaneously. PBLs were stimulated in vitro with rHBsAg and stimulation was expressed as stimulation index. Adoptive transfer of immunity to HBsAg was initially documented in 12 recipients of BM from anti-HBc+/anti-HBs+ donors. An almost 4 year follow-up showed detectable protective anti-HBs levels (>10 mIU/ml) in 50% of patients. Immunity to HBV was also documented in 22/35 BMT recipients (62%), who received their bone marrow from actively immunized donors. In 7/9 of these BMT recipients, anti-HBs antibodies levels were documented 25 months following BMT. In 6/8 (75%) of patients who received only PBLs from HBV immune donors, adoptive transfer of immunity to HBV, and seroconversion to HBsAg+, were documented within 2 months of i.v. injection. Evidence for specific cellular immune response with increased SIs was documented for healthy vaccinees, and BMT recipients, and in none of the healthy non-vaccinated controls. These results suggest that adoptive transfer of immunity to HBV is a useful method for providing long-lasting protection for BM recipients.

Adult↗

Induction of graft-versus-host disease and a graft-versus-leukemia effect using ubenimex in a patient with infantile leukemia relapsing after an unrelated cord blood transplant.

A 20-month-old boy with infantile leukemia was treated with total body irradiation, etoposide, cyclophosphamide and unrelated cord blood transplantation with a one-antigen mismatch. He relapsed on day 100 and achieved remission after ubenimex administration, and also developed chronic graft-versus-host disease of the skin. He remained in remission for 22 months with repeated courses of ubenimex. Ubenimex may be an alternative to donor lymphocyte transfusion and may be useful for the treatment of a patient who has relapsed after cord blood transplantation.

Cord Blood Stem Cell Transplantation↗

Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice.

BACKGROUND: CD23, the low affinity serum immunoglobulin E (IgE) receptor, is upregulated on B cells following interleukin (IL)-4 stimulation and is concomitantly cleaved to generate soluble CD23 (sCD23) fragments with cytokine-like activity. OBJECTIVE: Compounds that selectively inhibit the proteolytic release of CD23 to generate sCD23 were assessed for their ability to inhibit IgE production in order to evaluate the contribution of sCD23 in the production of human IgE as well as the ability of such compounds to block IgE production. METHODS: IgE production was measured in IL-4-stimulated human peripheral blood lymphocytes (PBL) and PBL-reconstituted SCID mice in the presence of a broad-spectrum matrix metalloprotease (MMP) inhibitor, a compound selective for inhibition of CD23 processing over MMPs and an anti-CD23 mAb, MHM6. RESULTS: The two compounds were equipotent in inhibiting IgE production without inhibition of IgG production by IL-4/anti-CD40-stimulated PBL. Soluble CD23 release was also shown to precede IgE accumulation in the cell-free medium. Addition of compound at later times other than day 0 in the 14 day assay resulted in progressively less inhibition of both IgE and sCD23, and exactly paralleled the effect of an anti-CD23 mAb, MHM6 on IgE levels. Both compounds also inhibited the release of CD23 from human RPMI 8866 cells adoptively transferred i. p. to mice. Doses required for inhibition of CD23 correlated well with the doses required for inhibition of IgE production in IL-4-challenged hu-PBL-SCID mice. IgE was selectively inhibited over total IgG in the SCID mice as well. CONCLUSIONS: Inhibition of CD23 processing alone is sufficient to inhibit IL-4-stimulated IgE production both in vitro and in vivo.

Animals↗

Ex vivo PKH26-labelling of lymphocytes for studies of cell migration in vivo.

A prerequisite for studies of cell migration is that the cells of interest can be appropriately labelled and subsequently easily traced. The use of radioisotopes or fluorescent substances that bind covalently to the cell surface, e.g. fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate (RITC), have limitations such as rapid loss of the labelling, toxicity and interference with cell surface molecules. In the present work the authors labelled rat spleen lymphocytes with the fluorescent labelling molecule PKH26, which is incorporated into the lipid bilayer of cytoplasmic membranes. The labelled lymphocytes were injected intravenously into syngeneic recipients and 2 or 6 days later the lymphocytes were detected in various organs by using flow cytometry and fluorescence microscopy. As could be expected, the lymphocytes homed to lymphoid tissues, preferably the spleen, and no labelled cells were found in non-lymphoid organs such as the heart and the kidney. Membrane labelling proved to be intense, uniform and stable and PKH26 positive cells were easily detectable in fractions less than 0.2% in peripheral blood and the various tissues after 6 days of in vivo circulation. Thus, the PKH26 dye appears to be suitable for labelling cell populations used in the study of cell migration in vivo, both under normal conditions and when specific immunological processes are taking place, such as graft rejection and tumour growth.

Animals↗

Divided we stand: tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester.

Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis.

Animals↗

Temporal changes in the distribution of thoracic duct lymphoblasts to synovium and other tissues of rats with adjuvant-induced arthritis.

The distribution of lymphoblasts(lymphocytes in cell cycle) obtained from the central lymph of donor rats and transferred adoptively to syngeneic recipients has been shown previously to be influenced by the presence of arthritis in either donor or recipient rats. The intent of the present study was to examine patterns of distribution of lymphoblasts in the early period after transfer, when extravasation of donor lymphoblasts was expected to occur. Thoracic duct lymphoblasts labelled in vitro with [125I]-iododeoxyuridine were detected in recipient rats by external radiometry and autoradiography. Irrespective of donor status, fewer donor lymphoblasts accumulated in the feet of normal recipients when compared to arthritic recipients at 15 min, 2 h and 24 h after cell transfer.When recipients of similar disease status were compared, the percentages of injected lymphoblasts from normal and arthritic donors recovered in the feet were similar at 15 min and 2 h after transfer. The proportions of lymphoblasts recovered in the feetat 24 h after injection declined in normal recipients and arthritic recipients of cells from normal donor rats. Importantly,this decline did not occur when both the donor and the recipient were arthritic. In the hindpaws, donor lymphoblasts were located predominantly in the bone marrow, except in transfers between arthriticrats, when at 24 h they were predominantly in the synovium. At 15 min, lymphoblasts were detected within the lumen of vessels within synovium, whereas by 2 h extravasation of these cells was evident. In conclusion, lymphoblasts accumulate more readily in hindfeet that are inflamed. In the early hours after injection, lymphoblasts from normal and arthritic donors are recruited equally, but these early levels are only maintained for 24 hin the combination of arthritic donor and arthritic recipient. Adramatic change in the proportion of lymphoblasts located in synoviumat this later time suggests that a dynamic process of relocation,retention and/or local cell division maintains the numbers of arthritic donor cells in the latter combination.

Adjuvants, Immunologic↗