Allan D. Hess

High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft-versus-host disease

Because of its potent immunosuppressive yet stem cell-sparing activity, high-dose cyclophosphamide was tested as sole prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation (alloBMT). We treated 117 patients (median age, 50 years; range, 21-66 years) with advanced hematologic malignancies; 78 had human leukocyte antigen (HLA)-matched related donors and 39 had HLA-matched unrelated donors. All patients received conventional myeloablation with busulfan/cyclophosphamide (BuCy) and T cell-replete bone marrow followed by 50 mg/kg/d of cyclophosphamide on days 3 and 4 after transplantation. The incidences of acute grades II through IV and grades III through IV GVHD for all patients were 43% and 10%, respectively. The nonrelapse mortality at day 100 and 2 years after transplantation were 9% and 17%, respectively. The actuarial overall survival and event-free survivals at 2 years after transplantation were 55% and 39%, respectively, for all patients and 63% and 54%, respectively, for patients who underwent transplantation while in remission. With a median follow-up of 26.3 months among surviving patients, the cumulative incidence of chronic GVHD is 10%. These results suggest that high-dose posttransplantation cyclophosphamide is an effective single-agent prophylaxis of acute and chronic GVHD after BuCy conditioning and HLA-matched BMT (clinicaltrials.gov no. NCT00134017).

Aldehyde Dehydrogenase Expression Drives Human Regulatory T Cell Resistance to Posttransplantation Cyclophosphamide

Regulatory T cells become resistant to cyclophosphamide after allogeneic stimulation, protecting against GVHD. Taming Graft-Versus-Host Disease Allogeneic bone marrow transplant is a curative therapy for many different diseases of the blood. Yet, the transplanted cells have a wild side—they sometimes can attack the recipient, causing graft-versus-host disease (GVHD). One way of preventing GVHD is with posttransplant cyclophosphamide, but what exactly cyclophosphamide does to the reactive T cells remains unclear. Now, Kanakry et al. report that regulatory T cells (Tregs) may play a critical role in this process. The authors observed that although cyclophosphamide treatment decreased conventional T cell numbers in patients, memory Tregs were relatively preserved. Tregs recovered faster than conventional T cells after therapy, in part due to the protective expression of aldehyde dehydrogenase (ALDH). Indeed, blocking ALDH in cultured cells sensitized Tregs to cyclophosphamide treatment. Moreover, in a xenograft model, removing Tregs prevented the protective effect of cyclophosphamide after transplant. These data provide insight into the mechanistic underpinnings of Tregs in transplant recipients. High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4+ T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4+ T cells with relative preservation of memory CD4+Foxp3+ T cells. In particular, CD4+CD45RA−Foxp3+hi effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4+Foxp3+ T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4+ T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy’s GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.

An in vitro predictive test for graft versus host disease in patients with genotypic HLA-identical bone marrow transplants

Acute graft versus host disease remains a major cause of morbidity and mortality in allogeneic bone marrow transplantation. To date, no clinically useful test has been reported that will predict the occurrence of graft versus host disease in genotypic HLA-identical donor-recipient pairs. We have developed a skin-explant model using donor lymphocytes that have been sensitized against recipient lymphocytes in vitro and cocultured with the recipients skin. Histologic changes compatible with acute graft versus host disease are found in the positive explants. To date 32 patients have been tested in a prospective manner. Among the 18 recipient-donor pairs that were positive, 16 patients were found to have histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Among the 14 negative pairs, only 3 patients had histologic Grade 2 or higher graft versus host disease of the skin on biopsy. Thus, the model has a sensitivity of 84 per cent and a specificity of 85 per cent, and is a significant predictor of the histologic occurrence of graft versus host disease (P less than 0.0005 by chi-square test). The test may be useful in the selection of donors for bone marrow transplantation and in the planning of prophylaxis against graft versus host disease.

Phase I trial of interferon gamma to potentiate cyclosporine-induced graft-versus-host disease in women undergoing autologous bone marrow transplantation for breast cancer.

PURPOSE We investigated if interferon gamma (IFN-gamma) could augment cyclosporine (CSA)-induced graft-versus-host disease (GVHD) following autologous bone marrow transplant in women with metastatic breast cancer and defined the toxicities of this therapy. PATIENTS AND METHODS Thirty-six women with advanced breast cancer were treated with CSA 2.5 mg/kg daily for 28 days and IFN-gamma 0.025 mg/m2 subcutaneously (SC) every other day, days 7 to 28 following autologous bone marrow transplantation and monitored for induction and severity of GVHD and toxicity of therapy. RESULTS GVHD was induced in 56% of patients. The severity of GVHD was greater than in a historic control population treated with CSA alone. Stage III rash was seen in 36% of patients, compared with 3% in the historic control population. Fourteen of 36 patients required therapy with topical corticosteroids and two of 36 required systemic treatment. Only three of 31 historic controls needed topical corticosteroids and no patient was treated systemically. There was no severe visceral GVHD. Hematopoietic recovery was not delayed. There were three toxic deaths. CONCLUSION CSA-induced GVHD can be safely augmented by IFN-gamma in women treated with high-dose alkylating agents and autologous bone marrow transplantation. There is little evidence of increased toxicity. Evidence of antitumor efficacy awaits further investigation.

Graft‐versus‐Host Reactions and Disease

Graft versus host reactions (GvHR) were initially a curiosity and graft versus host disease (GvHD) a frustrating problem to those interested in the clinical application of bone marrow transplantation (BMT) in various hutnan diseases. Clinical GvHD continues to be a probletn but its study in man and in animals, along with studies of GvHR, has come to be an exciting and fascinating area of research that continues to elucidate the principles and mechanisms involved in the control of the immune response to foreign antigens, alloantigens and to self antigens. This review is not intended to be comprehensive but rather an expression of our viewpoints derived largely from our own work in clinical and in laboratory investigations.

Treatment and prevention of acute graft-versus-host disease with thalidomide in a rat model

We have investigated the immunosuppressive effects of thalidomide (Thal) in a bone marrow transplant (BMT) model for graft-versus-host disease (GVHD). Lewis rats received RT1-incompatible marrow transplants from ACI rats after total-body irradiation. Twenty-two of twenty-three rats with established severe acute GVHD were successfully treated with Thal. Thal was given for therapy by gavage at 50 mg/kg/day or 100 mg/kg/day for 40 days after GVHD was clinically and histologically present. Fourteen of twenty-two received prophylaxis successfully with Thal at a dose of 50 mg/kg/day or 100 mg/kg/day. Acute GVHD did not develop after the drug was stopped. Three animals treated for severe GVHD later developed chronic GVHD. Chimerism was shown by permanent acceptance of ACI skin grafts and rejection of third-party skin grafts. Lymphocytes from Thal-treated animals likewise did not respond to Lewis or ACI cells in mixed lymphocyte culture but responded to third-party BN lymphocytes. Thal appears to be a potent new agent for therapy and prophylaxis of GVHD.

Suppressor cells in transplantation tolerance. I. Suppressor cells in the mechanism of tolerance in radiation chimeras.

Histoincompatible-complete radiation chimeras, after resolving acute graft-versus-host disease (GVHD), establish specific tolerance to host and donor alloantigens. This tolerance can be perturbed with immunosuppressive agents and infusions of small numbers of donor-type cells, with infusions of massive numbers of donor-type cells, or with infusions of a small number of donor-type cells, that were sensitized against host antigens prior to transfer. These chimeras possess T lymphocytes in the spleen that specifically suppress donor to host mixed lymphocyte reactions and adoptively transfer suppression of GVHD to secondary hosts. Nylon-wool fractionation of chimeric spleen cells restores the response of chimeric lymphocytes to host alloantigens, suggesting that transplantation tolerance is not attributable to clonal deletion but the activity of nylon-wool-adherent T suppressor spleen cells.

4-Hydroperoxyclophosphamide: a model for eliminating residual human tumour cells and T-lymphocytes from the bone marrow graft

Summary. Autologous bone marrow transplantation in acute leukaemia carries the risk of relapse from reinfusion of tumour cells present in marrow collected in remission and cryopreserved. An effective method for clearing marrow of tumour cells in required for a successful outcome. In the animal model 4‐hydroperoxycyclophosphamide (4‐HC) has proved to be effective in eliminating tumour cells from an autologous marrow graft. In the present studies, the in vitro effect of short‐and long‐term marrow cell incubation with 4‐HC on haemopoietic stem cells was investigated to determine the maximum concentration of 4‐HC that can be used for in vitro incubation without destroying the capacity of the marrow to effect complete haematological recovery as judged by residual CFUc content. However, loss of CFUc may not necessarily parallel survival of pluripotential stem cells. 4‐HC was also shown to be effective against peripheral T‐lymphocytes. Its possible therapeutic use in preventing or ameliorating graft‐versus‐host disease in allogeneic marrow grafts by preincubation with 4‐HC prior to transplantation is discussed.

C6 Produced by Macrophages Contributes to Cardiac Allograft Rejection

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.AaBu) cardiac allografts by PVG.1U (RT1.AuBu) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG.R8 (C6−) donors to PVG.1U (C6−) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6−) recipients of PVG.R8 (C6−) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6−) recipients that were reconstituted with bone marrow from PVG.1U (C6−) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Acute graft-versus-host disease: clinical characteristics in the cyclosporine era.

Graft-versus-host disease (GVHD) remains the major problem in allogeneic bone marrow transplantation. GVHD has limited the use of this technique to HLA-matched donor recipient pairs. Thus, only a quarter of patients who ultimately may have benefited from bone marrow transplantation are currently eligible. Even in matched patient recipient pairs, GVHD accounts for approximately 40% of the deaths following allogeneic bone marrow transplants. One of the major challenges for transplantation is to derive better strategies to prevent and treat GVHD while retaining the allogeneic benefit of graft-versus-leukemia. Current pharmacologic approaches have used cyclosporine, usually in combination with other drugs. More experimental approaches have removed lymphocytes from the marrow grafts. With either approach, maintaining the anti-leukemic benefit of an allogeneic transplant (i.e., immunologic attack of the leukemia resulting in a lower relapse rate), will need to be maintained if that approach will ultimately prove to be useful.