The interferonogenic agent tilorone as a modulator of GvH reaction in mice.

The effects of tilorone hydrochloride (TH), a powerful interferongenic agent exhibiting also immune modulatory properties on GvH reaction was studied, using the popliteal lymph node assay in mice. Administration of TH at days 0 or +2 relative to cell transfer to recipient mice led to a significant dose-dependent reduction of GvH reaction, whereas treatment of prospective donor mice at days-4 or-2 induced an enhanced GvH reactivity of donor spleen cells. This effect was found not to be due to an altered proportion in the spleen cell inoculum of B and T lymphocytes, the latter of which are responsible for induction of GvH reaction. However, since normal parental lymphocytes are prepared for an enhanced GvH reactivity by addition of TH-treated macrophages, a stimulatory effect of the latter cells via macrophage-derived mediators, induced by TH, is suggested.     

The role of danger signals and ectonucleotidases in acute graft-versus-host disease

Allogeneic hematopoietic cell transplantation (allo-HCT) represents the only curative treatment approach for many patients with benign or malignant diseases of the hematopoietic system. However, post-transplant morbidity and mortality are significantly increased by the development of acute graft-versus-host disease (GvHD). While alloreactive T cells act as the main cellular mediator of the GvH reaction, recent evidence suggests a critical role of the innate immune system in the early stages of GvHD initiation. Danger-associated molecular patterns released from the intracellular space as well as from the extracellular matrix activate antigen-presenting cells and set pro-inflammatory pathways in motion. This review gives an overview about danger signals representing therapeutic targets with a clinical perspective with a particular focus on extracellular nucleotides and ectonucleotidases.     

The role of tissue transglutaminase (transglutaminase type II) for the intestinal manifestations of murine semi-allogenic graft-versus-host disease

The intestinal manifestation of acute murine semi-allogenic graft-versus-host (GvH) disease is characterized by the occurrence of lymphocytic infiltrates in the lamina propria, by crypt hyperplasia and villous atrophy. In a histological respect, this animal model resembles human celiac disease. Tissue transglutaminase (tTG) (transglutaminase type II) has been identified to be the major B cell autoantigen in celiac disease. Furthermore, tissue transglutaminase has been implicated to be involved in its pathogenesis. Therefore, we aimed to investigate whether tissue transglutaminase is expressed in the intestines of GvH animals and whether its inhibition has any effect on the intestinal histology. Sera of patients with celiac disease and anti-tTG antibodies were purified. These antibodies were used for immuno-histochemistry of jejunal cryosection from GvH and syngenic control animals at day 6 after lymphocyte transfer. Furthermore, GvH mice were treated with antitTG antibodies and with the inhibitor of tissue transglutaminase monodansyl-cadaverine. The effect of this treatment on the development of crypt hyperplasia and villous atrophy were examined by light microscopy of hematoxylin-eosin (H&E) stained jejunal paraffin sections. We found a strong subepithelial expression of tissue transglutaminase in GvH animals but not in syngenic control mice. The localization of tTG seemed to be associated with the extracellular matrix (ECM). However, neither the treatment of GvH animals with anti-tTG antibodies nor the application of mono-dansyl-cadaverine prevented the development of crypt hyperplasia and villous atrophy. Similar to the situation in human celiac disease tissue, transglutaminase is highly expressed in the intestine of animals undergoing a semi-allogenic graft-versus-host reaction. However, this enzyme is probably not involved in the development of crypt hyperplasia and villous atrophy in this animal model.     

The antigen-inexperienced thymic suppressor cells: a class of lymphocytes in the young chicken thymus that inhibits antibody production and cell-mediated immune responses.

Transfer of thymus cells from young chickens in combination with a light whole body irradiation (360 R) was found to suppress the rejection of skin grafts across strong histocompatibility (B) differences. On the average, the suppressed animals also showed decreased serum hemagglutinin titers against erythrocytes of the skin donor strain and a decreased graft-versus-host (GvH) reactivity against embryos of this strain. The thymic suppressor cells can be obtained from animals that have not experienced the antigen under test. However, after transfer and contact to the antigen (skin graft) they can lead to the formation of specific ("activated") suppressor cells and can mediate in the long run a specific inhibition of the response to this antigen. The suppressive activity is associated with a bursa-dependent cellular subpopulation in the thymus that is different from B lymphocytes, B precursor cells or GvH-reactive T cells. The bursa dependency of the thymic suppressor cell suggests that functionally different lineages of thymic and thymus-derived lymphocytes are derived from different sources of prethymic stem cells. The suppressor cells are predominantly found in the young chicken thymus and already detectable in the 16-day-old embryo, while poor suppressive activity is found in the adult thymus. The suppressive effect can be obtained with thymus cells from either syngeneic or allogeneic donors. Embryonic allogeneic donors provide suppressive cell preparations free of GvH reactivity. The possibility that the thymus suppressor cells mediate self tolerance and "neonatal tolerance" is discussed.     

Graft-versus-host response in sex-linked dwarf, autosomal dwarf and control K strain chickens

Two independent experiments were conducted to examine the ability of 1) the autosomal-dwarf (ADW) strain and 2) the sex-linked (SLD)strain chicken to make a Graft-versus-Host (GvH) response. In each experiment the GvH response of the dwarf strain was compared to the GvH response of a normal growing control strain, the Cornell K strain chicken. All 3 strains were homozygous 15/ 15 at the major histocompatibility complex. GvH responsiveness of peripheral blood lymphocytes (PBL) from females of each strain was assessed at various intervals from 1.5 to 20 months of age using the chorio-allantoic membrane (CAM) assay.

The GvH response in female chickens from the ADW strain was significantly higher than in K strain females after the birds had reached sexual maturity (after 5.5 months). The GvH response in females from the SLD strain was, however, significantly lower than in the K strain females throughout the experiment. All strains tended to have a biphasic GvH response. There was a significant increase in GvH responsiveness from 1.5 to 5.5 months of age in chicks from all strains. In the SLD and K strain chickens, this was followed by a drop in the GvH response until 8.5 months of age. In 16-to 20-month-old SLD and K strain hens, the ability to mount a GvH response returned to levels observed in younger (5.5 months) pullets. The GvH responsiveness of the ADW strain remained at a constant level from 5.5 to 12 months of age. However, a second peak in GvH responsiveness was observed in 16-month-old ADW strain hens.

Strain differences in GvH responsiveness may be due to the known hormonal abnormalities in the dwarf strains. The suitability of these dwarf strains for the study of endocrine-immune interrelationships is discussed.     

Difference between the lymph node response to injection of autosensitized T lymphocytes and that in a graft-versus-host reaction

Thymus (T) lymphocytes autosensitized in vitro were shown in previous studies to produce enlargement of draining popliteal lymph nodes upon injection into the footpads of syngeneic rats. Specific autoreactive effector lymphocytes were found to be recruited within these lymph nodes. In the present study, the cellular basis of lymph node enlargement in mice by autosensitized lymphocytes was compared with that produced in a graft-vs.-host (GvH) reaction. T lymphocytes of C3H mice were autosensitized against syngeneic fibroblasts in vitro for 16 to 18 h in the absence of serum, and 10⁷ lymphocytes were injected into the footpads of syngeneic mice. Control lymphocytes were incubated without fibroblasts. The GvH reaction was produced by injecting 10⁷ C3H T lymphocytes into the footpads of (C3H × C57BL)F₁ adult recipients. The index of relative enlargement of the draining popliteal lymph nodes was measured 6 days after injection. Experiments were done to identify the origin of the lymph node cells in these reactions. Irradiation of the donor lymphocytes (1000 r) or the recipient mice (550 r) was used to prevent proliferation of the lymphocytes of either origin. The participation of recipient T lymphocytes in lymph node enlargement was investigated by using thymectomized mice. The following results were obtained. 1) The GvH lymph node enlargement was found to depend on proliferation of the donor T lymphocytes, but did not seem to require the participation of radiosensitive cells within the recipient mice. 2) In contrast, the response of the lymph nodes to autosensitized donor T lymphocytes depended on the function of radiosensitive T lymphocytes within the syngeneic recipients. The autosensitized donor lymphocytes themselves did not have to proliferate to recruit the response of recipient T lymphocytes. 3) It was found that recruitment of recipient lymph node cells could be super-imposed upon a conventional GvH reaction by presensitizing the C3H donor lymphocytes in vitro. Both autosensitization against syngeneic or allosensitization against C57BL fibroblasts augmented the lymph node response of (C3H × C57BL)F₁ hybrid recipients. The recruited or donor components of these mixed responses could be selectively abolished by irradiating either the donor lymphocytes or the recipient mice. Hence, the autosensitization response, like the host-vs.-graft transplantation reaction, can be induced by sensitization of lymphocytes peripherally and involves recruitment of lymphocytes within regional lymph nodes. The GvH response manifested in the same popliteal lymph nodes does not appear to require the recruitment of radiosensitive T lymphocytes. These findings suggest that different classes of T lymphocytes function in the autosensitization and GvH responses.     

Cellular composition of the chicken thymus; effects of neonatal bursectomy and hydrocortisone treatment

The cellular composition of the young chicken thymus has been analyzed by using 3 different physical parameters: cell volume, electrophoretic mobility, and the buoyant density of the cells. The analyses revealed the presence of 3 major subpopulations in the thymus of 16-week old chickens. These populations comprised about 66%, 19% and 15% of the thymic lymphoid pool. The data indicate that the last population, representing 15%, predominates in the medulla and is competent for GvH reactions. Marked effects on the cellular composition of the young chicken thymus are observed after neonatal bursectomy or after hydrocortisone treatment. Several findings suggest that the effect of bursectomy is not to be explained by B cell contamination in the chicken thymus. The data indicate the existence in the young chicken thymus of a bursa-dependent cellular subpopulation different from peripheral B lymphocytes but also different from cooperative or GvH active T cells. This subpopulation represents a part of the 66% small cells, and is estimated to constitute about 15% of the thymic lymphocyte pool.     

Augmentation of immune responses after methotrexate administration.

A single intraperitoneal injection of 0.5 mg methotrexate (MTX) has been found to increase the immune reactivity of spleen cells from (C57B1/6 X DBA/2)F1 mice. Five days after injection, spleen cells from MTX-treated mice exhibited greater PHA responsiveness and GvH reactivity, and mice given SRBC at this time developed greater than normal direct PFC responses. This pattern of effects of MTX was particularly evident in mice that had been given high doses of BCG intravenously 14 days before testing, a treatment that generally depressed the measured activities. MTX enhancement of GvH was also observed in mice that had been depleted of short-lived T lymphocytes by adult thymectomy. We suggest that MTX-sensitive cells possible exert, particularly in BCG-treated mice, a suppressive action on the responding cells.     

Acute Graft-versus-Host Reaction in SCID Mice Leads to an Abnormal Expansion of CD8+ Vβ14+ and a Broad Inactivation of Donor T Cells Followed by a Host-Restricted Tolerance and a Normalization of the TCR Vβ Repertoire in the Chronic Phase

The persistence and selection of allogeneic CBA/J T lymphocytes were studied during graft-versus-host (GvH) reaction in immunodeficient C. B-17 SCID (SCID) mice. After neonatal injection the donor cells primarily migrated to the spleen plus lymph nodes (SL) and the thymus of the recipients. Thirteen days post engraftment, CD8⁺ cells in SL had increased five times in cell number with an 18-fold increase of CD8⁺ Vβ14⁺ cells, paralleled by clinical signs of GvH disease (GvHD). Donor lymphocytes from these mice were proliferative unresponsive to allogeneic Balb/c or C57B1/6 SL cells, whereas 8 weeks post injection the tolerance was confined to H-2^d specific donor cells. Here, spleens had a total cell content similar to untreated SCID mice but the average percentage of donor cells had reached 25%. Moreover, the CD4/CD8 cell ratio in the donor population in SL and thymus had changed to normal and the TCR Vβ repertoire was similar to that of the originally injected cells. Following secondary transfer into syngeneic CBA/Ca nu/nu recipients donor cells regained a significant but reduced response to H-2^d stimulators indicating that the antigen specific tolerance of allogeneic donor cells in the SCID mice was due, at least in part, to a reversible state of anergy.     

Non-myeloablative stem cell transplantation for autoimmune diseases

Treatment of life-threatening autoimmune diseases in animal models with induced or spontaneous autoimmune diseases can be accomplished by a 2-step procedure involving elimination of self-reactive lymphocytes with an immune ablative conditioning regimen followed by infusion of autologous or allogeneic stem cells, respectively. In animal models it was shown that using such a strategy, autoimmunity could be adequately controlled. It is speculated that de-novo development of the T and B cell repertoire from uncommitted progenitor cells in the presence of the autoantigens may be the best recipe for re-induction of self-tolerance, similarly to the normal ontogeny of the immune system during the induction of self tolerance in fetal stage. For both autologous and allogeneic hematopoietic stem cell transplantation, a non-myeloablative stem cell transplantation (NST) regimen may be used for safer lymphoablation rather than myeloablation. In addition, for allogeneic hematopoietic stem cell transplantation engraftment of disease resistant donor stem cells will alter the genetic predisposition towards autoimmune disease susceptibility.     

Immunomodulation of GvH reaction in mice by Listeria monocytogenes

In the first part of experiments the GvHR activity of the spleen cell suspensions from normal parental donors (Balb/c) or infected with Listeria monocytogenes was compared. GvHR was examined in (Balb/c X AKR) F1 mice. Full suspensions from these donors or depleted of adherent cells or with addition of macrophages were studied. It has been proved that adherent cells population plays a significant role in the GvHR development. The addition of syngeneic macrophages from F1 hybrids results in a greater augmentation of GvH reactivity of parental lymphocytes. However, the addition of macrophages harvested from F1 hybrids immunized with L. monocytogenes 7 days before, brings about a weaker GvH reaction. Spleen cells of parental donors injected with L. monocytogenes 5-6 days earlier induced stronger GvHR as compared with the splenocytes of normal donors. Similarly, F1 hosts treated with L. monocytogenes 3-5 days before injection of normal donor's spleen cells showed increased GvHR, but those infected 7-9 days before injection of spleen cells, developed only very weak GvHR.     

Inhibition of T cell activity in vivo: a test model for quantitative evaluation.

A test model is presented which, in comparison with the conventional models of skin transplantation or graft-versus-host (GvH) reaction in mice, permits a more sensitive quantitative evaluation of T cell inhibition in vivo. Prospective donors (type AA) are immunized with prospective recipient material (type AB); the resulting T cell reaction of A versus B is inhibited by consecutive treatment. Extent of inhibition can be evaluated after transfer of the pretreated AA material onto AB recipients by calculation of remaining GvH reactivity, if compared to adequate control tranfers. In this model the target animal for T cell reactivity (the AB recipient) remains untouched from immunosuppressive regimen.     

The Host Component of the Popliteal Lymph Node Graft-versus-Host Reaction

We have examined the cellular changes taking place in rat popliteal lymph nodes undergoing a graft-versus-host (GvH) reaction. Examination of immunoperoxidase-stained lymph node sections, using a panel of mouse monoclonal antibodies directed against different rat lymphoid cell subsets, revealed a disorganization of the lymph node architecture with disappearance of the follicles, and an intermingling of T and B cells, so that no distinct T- and B-cell areas were visible any more. Since the GvH nodes showed a preferential accumulation of host B cells over host T cells (particularly over the W 3/25+ T helper cell subset), we also investigated the requirements for host B cell activation. The popliteal lymph node GvH reaction was induced in (PVG X DA)F1 rats by the injection of PVG cells into one foot and by DA cells into the other foot, and then immunoglobulin kappa allotype marked PVG B cells from athymic donors were injected intravenously. The allotype marked B cells proliferated vigorously in response to the DA T cells, but much less in response to the PVG T cells. These results indicate that the massive B-cell activation taking place in GvH reactions may require an alloantigen incompatibility between donor T cells and host B cells, and argue against non-specific mitogenic induction of the B cells.     

CD43 is expressed normally on Wiskott-Aldrich-derived lymphocytes.

The Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by eczema, thrombocytopenia, and profound immunodeficiency in affected males. While the etiology of the syndrome is currently unknown, abnormalities of CD43 have been described as a biochemical marker of the disease. Several investigators have demonstrated alterations in the expression of the CD43 surface antigen on WAS hematopoietic cells, noting either absence, decreased levels or changes in the characteristic molecular weight of the protein on the lymphocytes of affected patients. Biochemical studies have further indicated that glycosylating activity of specific enzymes which may post-translationally modify CD43 is altered in both T cells and Epstein-Barr-virus (EBV)-transformed B cells in WAS patients when compared to unaffected controls. Here we present data on cells derived from two males with a clinical diagnosis of WAS. Analysis of genomic DNA from the mothers of each of these patients (obligate carriers) showed a nonrandom X-chromosome inactivation pattern of nucleated blood cells, confirming the diagnosis of the X-linked syndrome. CD43 was characterized on peripheral blood lymphocytes and long-term EBV-transformed B cell lines, both to further analyze the molecular defects of WAS, as well as to attempt to generate a reproducible method for disease detection. Surprisingly, surface expression, molecular weight and two-dimensional gel analysis failed to demonstrated any reproducible differences in the CD43 expression, whether from disease or normal lymphocytes. Such results suggest possible heterogeneity of this syndrome.     

Stimulation of regulatory T cell circuits by immunoglobulin-dependent structures on activated B cells

An immunoregulatory circuit is described in which B cell blasts activate syngeneic Ly-1⁺2^?3^? T cells to (a) start a reaction which is indistinguishable from a graft-vs.- host reaction (syngeneic GvH) and (b) induce suppressor cell activity which abrogates the syngeneic GvH. Since capping the surface immunoglobulin (Ig) on B cell blasts blocks their ability to activate this circuit, it is likely that the relevant cell surface structure “seen” on the B cell by the Ly-1 T cell is either Ig itself or another molecule in association with Ig.     

Dominance and persistence of donor marrow in long-lived allogeneic radiation chimeras obtained with unmanipulated bone marrow

Allogeneic, H-2-incompatible irradiation chimeras (H-2d leads to H-2b) constructed with normal, unmanipulated bone marrow and with marrow-derived factors live long and do not manifest a GvH disease. Their response to primary immunization is deficient but their alloreactivity is normal. This chimeric allotolerance cannot be passively transferred from chimeric donors to normal irradiated recipients. Passive transfer of both donor- or recipient-type immunocompetent T-cells into the chimeric mice does not lead to syngeneic reconstitution, rejection of the engrafted marrow or GvH disease and the mice maintain permanently their chimerism. This new model demonstrates that chimerism is not eradicable in long-lived chimeras reconstituted with unmanipulated bone marrow, and that the bone marrow itself plays a dominant role in maintenance of chimerism.     

Studies on the anti-inflammatory and immunotropic effect of gold salts

The experiments carried out in vitro and in vivo aimed at evaluation of the effect of the selected gold salts on the experimental inflammatory reactions and cellular immune reactions, specific and nonspecific. The preparations investigated appeared to inhibit relatively weakly nonspecific inflammatory processes (granulation test, lysozym level) but to strongly inhibit the cellular immune reactions (GvH reaction, LNPF test, contact hypersensitivity). The possible mechanisms of the gold salts effect are discussed which were shown neither to consist in the stabilization of cell membranes nor to act through the thymus factors or the influence on quantitative relations between T and B lymphocytes. A suggestion has been put forward that gold salts exert direct effect on the mature final cells participating in the immune processes.     

Specific suppression of the local GVH reaction by F1 spleen cells

The local graft-versus-host (GvH) reaction in (C57BL/6 X BALB/c) F1 hybrid mice, assayed by popliteal lymph node enlargement, was specifically depressed by an injection of parental lymphocytes mixed with spleen cells from F1 mice pretreated with the same parental lymphocytes. Suppressor activity of CBF1 spleen cells was obtained 7 days after inoculation of parental lymphocytes, and peaked on day 10. The suppressive activity was induced by only spleen cells from CBF1 which was inoculated Balb/c lymphocytes, but not C57BL/6 lymphocytes. The lymphocyte subpopulation responsible for the suppressive activity was noticed in T cell population.     

Treatment of donor cells with l-leucyl-l-leucine methyl ester prevents induction of graft-vs-host-like reaction in [lprlpr → +/+] chimera

Irradiated C57BL/6 (B6) mice which had received spleen cells from autoimmune-prone C57BL/6J-lpr/lpr (B6-lpr) mice underwent a graft-versus-host (GvH)-like reaction early after the spleen cell transfer, although both strains have the same background genes, including MHC and Mls gene, but differ only in a lpr gene. We analyzed the changes in this GvH-like reaction when the donor spleen cells had been treated with l-leucyl-l-leucine methyl ester, which has been reported to have an inhibitory effect on the early GvH reaction in allogeneic or semiallogeneic chimeras. The treatment of donor spleen cells completely abrogated the induction of the early phase of the GvH-like reation in [B6-lpr → B6] chimeras. The results suggest that the GvH-like reaction in these chimeras is caused by a mechanism(s) similar to that operating in allogeneic or semiallogeneic chimeras.