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CD4+CD25+ regulatory T cells (T(REG)) are engaged in the regulation of murine and human immune responses as well as graft-versus-host disease (GvHD) after allogeneic stem-cell transplantation. Despite their suppression of GvHD they do not impair graft-versus-tumor activity in the mouse, which makes T(REG) especially attractive candidates for cellular immunotherapy. T(REG) comprise only 5% to 10% of CD4+ T cells in peripheral blood and are naturally anergic, which prevented their use as therapeutic suppressor cells in the context of autoimmune or alloimmune reactions so far. We therefore developed an in vitro expansion protocol for human T(REG), breaking their anergy with anti-CD3/anti-CD28-coupled paramagnetic beads and a combination of interleukin (IL)-2 and IL-15. Highly purified human T(REG) can be expanded 285-fold to 1000-fold within 20 days and keep their phenotype as well as all their suppressor functions even in the context of stimulation with mature allogeneic dendritic cells. However, we demonstrate that FoxP3 is not a reliable marker for human T(REG) as it is transiently inducible in CD4+CD25- cells upon activation with cytokines or via their T cell receptor. In addition, we successfully expanded CD4+CD25+ cells from patients after allogeneic stem-cell transplantation with or without GvHD and show that different suppressor functions might be lost independently, demonstrating that human T(REG) biology is likely more complicated than previously thought.

Background: The use of monoclonal antibodies against interleukin-2 receptor (IL-2R)-alpha chains could be an effective treatment of acute graft-versus-host disease (GvHD). Experimental model and clinical studies have reported various results., Methods: Inolimomab is a murine anti-IL-2R. Eighty-five patients were evaluated retrospectively for the safety and efficacy of inolimomab given for the treatment of steroid-resistant acute GvHD (aGvHD) following allogeneic hematopoietic stem cell transplantation (HSCT). Diseases were immune deficiency, hematological malignancies, or solid tumors. Seventy-six percent of the patients received a myeloablative regimen. The source of HSCT was bone marrow for 45 patients, peripheral blood for 36 patients, and cord blood for 4 patients. Donors were 49 siblings and 36 unrelated. Acute GvHD was diagnosed within a median of 28 days after transplantation (grade II, 26 patients; grade III, 26 patients; grade IV, 33 patients). Inolimomab was administered in the event of steroid-resistant aGvHD with a median dose of 0.468 mg per kg (median period of treatment: 18 days)., Results: Twenty-five complete responses and 29 partial responses (total response rate: 63%) were observed with no side effects. There was no correlation between aGvHD grading and quality of response. Better responses were observed in cutaneous aGvHD. The overall survival probability was 26% (median follow-up: 20 months). Fifty-seven percent of patients died of toxicity related mortality, mostly aGvHD. Response to inolimomab seemed sustained (11% relapse in responders)., Conclusion: Inolimomab is well-tolerated and effective for severe steroid-resistant aGvHD. The optimum regimen remains to be defined.

Despite prophylaxis with immunosuppressive drugs, severe graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality following allogeneic stem cell transplantation. T cell depletion of the graft has decreased incidence and severity of GVHD but has resulted in a higher incidence of graft failure and relapse. To reduce the risk of severe GVHD, but at the same time to maintain the graft-versus-tumor effect, we administered a fixed low number of 1 x 10(5) donor T cells per kilogram of body weight to recipients of CD34(+) cell-enriched allogeneic peripheral blood stem cells (PBSCs). Donor lymphocyte infusions (DLI) were then given in incremental doses when mixed chimerism persisted or signs of relapse occurred. A total of 23 patients receiving allografts from related and unrelated donors were treated according to this protocol after myeloablative or reduced intensity conditioning. One patient did not engraft and 3/20 evaluable patients developed acute (a) GVHD > or = grade II, with a corresponding estimated cumulative incidence of 15.6% (95% CI 0-30.5%). DLI (n = 13) induced aGVHD > or = II in 6 patients, but the severity of the syndrome was reduced. Overall GVHD-related mortality was low (13%). The probability of disease-free survival and overall survival at 2 years was 0.40 (95% CI 0.21-0.75) and 0.36 (95% CI 0.21-0.63). Progression-free survival and overall survival was significantly better in patients with acute or chronic myelogenous leukemia compared to patients with lymphoproliferative disorders (p = 0.002; p = 0.02). We conclude that the combination of allografts with a T cell content of about 1 x 10(5)/kg and escalating doses of DLI is a viable transplant strategy in patients with myeloid leukemias, which results in stable engraftment and a reduction of mortality from aGVHD.

We analyzed effects of successive changes in prevention and treatment of chronic GVHD in 405 patients with aplastic anemia and refractory anemia given HLA-matched hematopoietic stem cell transplantation (HSCT) from 1970-1997. For analysis, patients were divided into group I, transplantations from 1970-1976; group II, 1977-1983; group III, 1984-1990; and group IV, 1991-1997. The overall incidence of chronic GVHD was 28%. Incidences of chronic GVHD for groups I through IV were 20%, 46%, 41%, and 22%, respectively, reflecting added buffy coat infusions in groups II and III. Five-year survival rates of patients with chronic GVHD for groups I through IV were 58%, 74%, 82%, and 76%, respectively (NS). Among group I patients, 50% were alive off immunosuppression, none were alive on immunosuppression, and 50% died. These figures were 76%, 0%, and 24% in group II; 80%, 10%, and 10% in group III; and 64%, 21%, and 14% in group IV patients. More serious infections and skin contractures were seen in group I than in groups II, III, and IV (P = .0001, .02, .01 and P =.0003, .001, .05, respectively). Lung complications, aseptic necroses, depression, and Karnofsky scores were comparable among groups. Gastrointestinal complications seemed less frequent among groups II through IV. Diabetes mellitus was more frequent in group IV than in groups I through III (P = .008). Secondary malignancies occurred in 33%, 6%, 3%, and 0% of patients in the 4 groups, respectively. In conclusion, over 28 years, chronic GVHD has remained challenging, with only slight improvements in quality of life. Decisive improvements in therapy and survival will have to await both a better understanding of the immunological events underlying chronic GVHD and better infection prevention and control.