Allogeneic Reconstitution Research Articles

Cytokines in lethal graft-versus-host disease.

Graft-versus-host disease (GVHD) is caused by donor T lymphocytes that recognize foreign antigens on host tissues. This leads to T cell activation, which involves a cascade of events including the transcription of genes for cytokines and their receptors and the production of cytokines. One of the first cytokines to appear is interleukin 2 (IL-2). IL-2 production enhances the IL-2 receptor expression and leads to T cell proliferation. As a further step, differentation of T cells occurs, which results in the production of a certain pattern of cytokines. These cytokines influence the expression of cell surface antigens and adhesion molecules, and are able to activate other cell types such as cytotoxic T cells, macrophages and natural killer cells, which might act as effector cells in tissue destruction. Insight into the sequential expression of the various cytokines involved might enable a more effective treatment of GVHD. Therefore, we investigated the occurrence of cytokines in a murine model for acute GVHD. We addressed in particular the period early after allogeneic reconstitution.

MIXED ALLOGENEIC RECONSTITUTION (A+B→A) TO INDUCE DONOR-SPECIFIC TRANSPLANTATION TOLERANCE

Mixed allogeneic reconstitution, in which a mixture of T-cell-depleted bone marrow of syngeneic host and allogeneic donor type is transplanted into a lethally irradiated recipient (A+B----A), results in mixed lymphopoietic chimerism with engraftment of a mixture of both host and donor bone marrow elements. Recipients are specifically tolerant to donor both in vitro and in vivo. Donor-specific skin grafts survive indefinitely when they are placed after full bone marrow repopulation at 28 days, while third-party grafts are rapidly rejected. To determine whether a delay of a month or more for full bone marrow repopulation is required before a donor-specific graft can be placed, we have now examined whether tolerance induction can be achieved if a graft is placed at the time of bone marrow transplantation. Permanent acceptance of donor-specific B10.BR skin grafts occurred when mixed allogeneic chimerism (B10+B10.BR----B10) was induced and a simultaneous allogeneic donor graft placed. In vitro, mixed reconstituted recipients were specifically tolerant to the B10.BR donor lymphoid cells but fully reactive to MHC-disparate third-party (BALB/c; H-2dd) when assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) assays. These data therefore indicate that a donor-specific graft placed at the time of mixed allogeneic reconstitution is permanently accepted without rejection. To determine whether an allogeneic skin graft alone without allogeneic bone marrow would be sufficient to induce tolerance, syngeneic reconstitution (B10----B10) was carried out, and a simultaneous B10.BR allogeneic skin graft placed. Although skin grafts were prolonged in all recipients, all grafts rejected when full lymphopoietic repopulation occurred at 28 days. Taken together, these data suggest that allogeneic donor bone marrow elements are required for the induction and maintenance of donor-specific transplantation tolerance and that allogeneic skin grafts alone are not sufficient for tolerance induction.

Photoinactivation of T-Cell Function with Psoralen and UVA Radiation Suppresses the Induction of Experimental Murine Graft-Versus-Host Disease Across Major Histocompatibility Barriers

Bone marrow transplantation is employed in the treatment of a number of hematologic and malignant diseases. A major complication is the induction of graft-versus-host disease. Whereas removal of T lymphocytes from the donor marrow effectively reduces the incidence of graft-versus-host disease, the incidence of graft failure often increases when T cells are depleted from the transplanted marrow. In the current study, photoinactivation of the donor cells with 8-methoxypsoralen coupled with exposure to long-wavelength ultraviolet radiation (PUVA therapy) was used to inactivate the response of the donor T cells against the host. PUVA therapy suppressed the ability of spleen cells to respond to alloantigen in the in vitro mixed lymphocyte reaction. The induction of acute graft-versus-host disease across complete major histocompatibility barriers in lethally X-irradiated mice was significantly suppressed after bone marrow transplantation with photoinactivated bone marrow cells. Long-term survivors demonstrated allogeneic reconstitution and partial restoration of T-cell function. Because PUVA therapy had no inhibitory effect on hematopoiesis, these data suggest that using phototherapy to inactivate the alloreactivity of T cells may provide an alternative to purging T cells from the donor marrow, thus suppressing both the incidence of graft-versus-host disease and the incidence of graft failure.

In vitro and in vivo analysis of bone marrow-derived CD3 +, CD4 −, CD8 −, NK1.1 + cell lines

The development of methods of avoiding graft-versus-host disease (GVHD) while retaining the alloengraftment-promoting and anti-leukemic effects of allogeneic T cells is a major goal of research in bone marrow transplantation (BMT). We have recently obtained evidence suggesting that natural suppressor (NS) cells derived from T cell-depleted (TCD) syngeneic marrow can protect against GVHD while permitting alloengraftment. We have now attempted to enrich and then propagate NS cells in vitro, with the goal of obtaining an enhanced anti-GVHD effect by adoptive transfer in vivo. Two long-term cell lines were generated culturing BMC depleted of Mac1-positive cells and of Mac1-positive plus Thy1-positive cells in high concentrations of IL-2. Both cell lines showed anti-GVHD effects when administered along with a GVHD-producing inoculum, while permitting complete allogeneic reconstitution. A clone derived from Mac1-depleted BMC protected completely against a more chronic pattern of GVHD. These cell lines demonstrated suppressive activity in vitro, cytolytic activity against a broad range of natural killer (NK)-sensitive and NK-resistant targets, and a novel cell surface phenotype, with characteristics of both αβ-TcR-bearing T cells and of NK cells. In some respects, these cells resemble LAK cells and differ from fresh NS cells, and from the cloned NS cells derived from spleens of total lymphoid irradiation (TLI)-treated mice and neonatal mice. To our knowledge, this is the first detailed phenotypic analysis of cell lines with in vivo anti-GVHD activity. If applicability can be demonstrated in large animal models, the ability to use bone marrow as a source of such protective cell lines might also have potential utility in clinical BMT.

In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment.

Previous work from this laboratory has demonstrated that T cell-depleted (TCD) syngeneic marrow can delay, but not prevent, the mortality from acute graft-vs.-host disease (GVHD) caused by MHC-mismatched lymphoid cells administered to lethally irradiated mice. We demonstrate here that a protective effect against GVHD is also observed after in vivo treatment with IL-2. Administration of 10,000-50,000 U of IL-2 twice daily for the first 5 d after bone marrow transplantation markedly reduced the mortality from both acute and chronic GVHD induced across complete MHC barriers in lethally irradiated mice, and frequently led to long-term survival. Complete allogeneic reconstitution was demonstrated in all long-term survivors of this treatment regimen. While either IL-2 or TCD syngeneic marrow administered alone was protective in some experiments, the maximal protective effect was observed after administration of both IL-2 and TCD syngeneic marrow, especially when the effects of IL-2 were suboptimal. The timing of IL-2 administration was critical to this beneficial effect, since a delay of 7 d in commencing IL-2 treatment was associated with accelerated GVHD mortality. This new approach to the prevention of GVHD permits the administration of allogeneic T cells, and may therefore avoid the increased incidence of graft failure and loss of antileukemic effects associated with the T cell depletion of allogeneic marrow, which is otherwise required for the prevention of GVHD.

Alloresistance to engraftment of allogeneic donor bone marrow is mediated by an Lyt-2+ T cell in mixed allogeneic reconstitution (C57BL/10Sn + B10.D2/nSn----C57BL/10Sn).

In the mixed allogeneic reconstitution (B10 + B10.D2----B10) model, alloresistance to engraftment of allogeneic donor results if the syngeneic component of the mixed bone marrow inoculum is not depleted of Lyt-2+ cells before transplantation. Resultant experimental animals repopulate as fully syngeneic, reject B10.D2 skin allografts, and are reactive to B10.D2 lymphoid cells in vitro, as assessed by mixed lymphocyte culture proliferative and cellular cytotoxicity assays. In contrast, depletion of Lyt-2-reactive cells from the syngeneic component of the mixed bone marrow inoculum results in mixed lymphopoietic chimerism and specific in vivo transplantation tolerance to B10.D2 allogeneic donor skin grafts and in vitro unreactivity to B10.D2 lymphoid elements. Full reactivity to third party is evident both in vitro and in vivo in these animals. This model may be helpful in further study of the syngeneic host-type cell phenotypes responsible for alloresistance to bone marrow engraftment.

Changes in Ia expression in mouse kidney during acute graft-vs-host disease.

We induced graft-vs-host disease (GVHD) in mice to determine whether immunologic stimuli could alter renal Ia expression. Two strain combinations were used: B6.C-H-2bm12 into C57BL/6, an I-A mutation difference, and A.SW into A.TL, differing in the I and D regions of H-2. By day 10 after allogeneic reconstitution of lethally irradiated recipients with bone marrow and spleen cells, the recipients had developed acute GVHD, as measured by their spleen to body weight ratio. Histologic examination revealed focal interstitial infiltrates of mononuclear cells in the kidneys. The expression of host Ia in these kidneys was increased up to 10-fold, as measured by absorption, and indirect immunofluorescence indicated that certain renal tubule cells had become strongly positive, suggesting that these were the principal sites of the increase in Ia expression. Similar increases were not observed in donor Ia. Tubule cells may have become Ia positive by passive uptake, or more probably, by the increase of Ia biosynthesis in cells that usually synthesize little or no Ia. Lethal irradiation without reconstitution tended to decrease renal Ia expression, as assessed by absorption and immunofluorescence. The results indicate that renal Ia expression, particularly in renal tubules, can be altered by changes in the immune system, raising the possibility of a role for such altered Ia expression in autoimmune or alloimmune responses involving the kidney.