ALBERT

Description: Pre-clinical data in monkeys receiving non-myeloablative conditioning followed by MHC-mismatched kidney and bone marrow transplantation show that transient chimerism is sufficient to permit achievement of long-term tolerance to a simultaneous donor renal allograft. Our group has recently reported successful induction of tolerance to donor kidneys in patients with advanced multiple myeloma and renal failure through combined bone marrow and kidney transplantation in the HLA-identical setting. On the basis of these results, five end-stage renal failure patients without malignant disease received simultaneous kidney and bone marrow transplantation from haploidentical HLA mismatched related donor after non-myeloablative conditioning with MEDI-507 (anti-CD2 humanized mAb; MedImmune), cyclophosphamide, thymic irradiation and peritransplant cyclosporine. Transplantation of kidney and bone marrow were both performed on Day 0. All patients developed initial mixed chimerism but lost their chimerism by Day 21. We have analyzed three patients who successfully discontinued immunosuppression on Days +240, +422 and +244. At a follow-up of 47, 38, and 17 months, all three patients are off immunosuppression without allograft rejection. T-cell counts exceeded 100 cells/μL by Day +128, +21, +21, respectively, and a high early prevalence of CD4+CD25high cells was detected. Post-transplant in vitro alloreactivity assays (bulk MLR/CML) showed the development of long-lasting donor-specific unresponsiveness in all three patients. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, no killing of donor RTEC was detected post-transplant. We also assessed alloresponses in chemorefractory lymphoma patients receiving haploidentical bone marrow transplantation with a similar conditioning regimen. In contrast to the recipients of combined kidney/bone marrow transplants, these patients showed sustained global hyporesponsiveness in CML and MLR. However, loss of donor chimerism was associated with the eventual appearance of measurable anti-donor CML and/or MLR responses. In contrast, donor-specific and host-specific unresponsiveness with strong anti-3rd party responses developed in a sustained mixed chimera who received haploidentical stem cell transplantation with a modification of this conditioning protocol (i.e. different dose of MEDI-507, Isolex-selected CD34+ cells from G-CSF mobilized PBMC and the addition of fludarabine). In summary, we have obtained proof of principle that durable multilineage mixed chimerism with donor- and host-specific tolerance can be achieved without GVHD in humans receiving haploidentical HCT. In recipients of combined kidney/bone marrow transplants but not in recipients of bone marrow alone, patients who lose chimerism develop donor-specific unresponsiveness. These studies point to a role for the renal allograft in maintaining long-term tolerance following loss of initial donor chimerism.

Description: Abstract 3956 Background: Recently in large randomized studies, the addition of Rituximab to standard chemotherapy in the first-line treatment of follicular lymphoma (FL) demonstrated improvements in long-term outcome in FL. Methods: We compared the outcome of 111 naive FL patients (pts) treated at our institution from 1995 to 2009 with a single alkylating agent in combination or not with Rituximab: 58 pts received Chlorambucil plus Rituximab (R-Chl) and 53 pts Chlorambucil and prednisone (Chl+PDN). The 2 schedules included an induction phase, where Chlorambucil was given at 6mg/mq for 6 consecutive weeks in both groups and the Rituximab in 4 weekly administrations. The maintenance phase was longer in the Chl+PDN group: Chlorambucil was administered 6mg/mq daily 14 days each month for a total of 12-months of treatment versus 14 days with monthly-Rituximab for 4 consecutive months in the R-Chl group. Results: The demographic and prognostic factors are reported in Table 1. At the end of treatment the ORR was 98% in the R-Chl pts and 77% in the Chl+PDN group, with a percentage of complete response of 79% and 55% respectively. No significant incidence of adverse events were reported and only one HBsAg positive patient in R-Chl group discontinued the therapy. One case of myelodysplastic syndrome was described in a Chl+PDN relapsed patient. With a median follow up of 34 months, the OS and the EFS in R-Chl pts is 95% and 76% respectively. Otherwise, with a median observation time of 82 months in the Chl+PDN group 73% are alive and only 28% of pts maintained the response. The median time to subsequent therapy (TTST) is 21 and 15 months respectively in the 2 groups. Conclusions: The addition of Rituximab to alkylating in first-line treatment in previously untreated FL pts improves significantly the EFS and prolongs the time to next antilymphoma therapy compared with alkylating alone. Because of its low toxicity profile, our combination with Rituximab and Chlorambucil could be considered a first-line therapy, especially in FL patients not eligible for aggressive chemotherapy regimens. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4700 Acute and chronic graft-versus-host disease are a common complication of allogeneic stem cell transplantation. In animal models acute GVHD (aGVHD) is associated with increased neovascularization and number of circulating endothelial cells (CECs), while patients with sclerodermatous chronic GVHD (cGVHD) show a significant decrease in the number of circulating endothelial progenitor cells (EPCs) in peripheral blood as compared to patients with non sclerodermatous cGVHD or controls. In an attempt to evaluate the role of CECs and EPCs in patients with cGVHD, we analysed a total of 15 patients affected by hematological malignancies (3 Non-Hodgkin Lymphoma, 4 Hodgkin Disease, 4 Multiple Myeloma, 2 Myelodisplastic Syndrome, 2 Chronic Lymphocitic Leukemia) having undergone allogeneic stem cell transplantation following reduced intensity conditioning. Donors were HLA identical in 14 patients (12 sibling and 2 matched unrelated donors) and HLA aploidentical in 1. Acute GVHD and cGVHD were defined on the basis of time of manifestation, ≤100 days for aGVHD and 〉100 days for cGVHD. At the time of the blood sample collection, 8 patients, median age 42 years (28-51), with a median time after transplant of 177 days (21-1373), had no evidence of GVHD; of those 5/8 were evaluable for aGVHD and cGVHD, 2 only for aGVHD; 4/8 were on post-transplant calcineurin inhibitors immunosuppressive therapy; 7 other patients, median age 51 years (38-64), with a median time after transplant of 844 days (314-1779), were all evaluable for acute and cGVHD and had evidence of cGVHD as follows: sclerodermatous in 3 patients requiring systemic immunosuppressive therapy, oral mucosa lichen in 1 patient taking oral corticosteroid and cutaneous erythematous and dischromic cGVHD in the other 3 patients, with only 1 patient on systemic immunosuppressive therapy. Viable and apoptotic CECs and EPCs were evaluated by six color flow cytometry (Mancuso et al, Clin Cancer Res, 2009). Briefly, CECs were defined as DNA+CD45-CD31+ CD146+, EPCs as CD45- CD34+. The combination of Syto16 and 7-AAD was used to discriminate between viable (syto16bright/7-AAD-) and apoptotic (syto16weakly pos/7-AAD+) endothelial cells, and to exclude from analysis, platelets and endothelial macroparticles. The results, expressed as median of cells/mL, are summarized in the following table:Total CECsViable CECsApoptotic CECsEPCsHealthy Subject103 (33–322)21 (3–67)77* (28–303)31 (0–56)Patients with cGVHD46 (29–94)41 (25–68)5* (3–26)30 (0–213)Patients without GVHD138 (30–179)39 (10–153)68* (5–136)49 (0–355)*p

Description: DLBCL can be divided into two prognostically different subgroups, germinal center B-cell-like (GCB) and activated B-cell-like (ABC), according to twodifferent gene patterns identified with cDNA microarray technology. Though valuable, this technology is expensive and not generally available. However, the identification of individual antigens related to different stages of B-cell differentiation using immunohistochemistry, can be used to assess the two profiles yielding comparable results with respect to cDNA microarray technique. We have retrospectively investigated 105 patients (pts) diagnosed with de novo DLBCL and treated at our centre between November 2001 and June 2004; the only inclusion criteria was the availability of a tissue biopsy at diagnosis. Median age was 62 (19–85); stage at diagnosis was I–II in 49 pts (47%), III in 14 pts (13%) and IV in 42 pts (40%); according to IPI, 74 (70%) pts were defined as low (0–2) and 31 (30%) as high risk (3–5). Interestingly, the majority (53%) of our pts, had a primary extranodal lymphoma at diagnosis. TMA analysis was performed with antibodies to CD10, bcl-6 and MUM1 allowing the following classification: 50 pts (48%) were considered having CGB lymphoma and 55 pts (52%) having ABC disease. According to IPI risk score, 38 pts with CGB and 36 with ABC lymphoma were at low risk (0–2) whereas 10 with CGB and 16 with ABC lymphoma were at high risk (3–5). All pts received a median of 6 cycles of a CHOP-like, antracycline-based polychemotherapy. Observed ORR was 89% (94/105); 62 (59%) pts achieved a CR, 32 (30.5%) a PR while 11 (10.5%) failed to respond to treatment. Median follow-up of the surviving pts was 45 months ( 5–110). The 3-year OS for the entire group was 71% and the 3-year EFS was 54%. In terms of CR, PD and resistance to therapy, no difference was observed between the two TMA types of DLBCL. Pts obtaining a CR after 1st line treatment were equally distributed in both groups (28.6% in CGB vs 30.5% in ABC) as were those not responding to therapy (3.8% in CGB versus 6.7% in ABC). A separate analysis in pts with stage IV disease at diagnosis was also performed and showed similar results (40.5% of CR, 23.8% of NR; no difference was observed between the two TMA defined subgroups). Pts who experienced a PD at any time, were equally distributed between the 2 subgroups, either if they relapsed after first line therapy (6 pts in CGB group , 10 in the ABC one) or after any other subsequent treatment (12 in the CGB group, 18 in the ABC one). Furthermore, 39 of 56 (69%) patients with extranodal presentation at diagnosis showed a CR independently of their subgroup distribution (37.5% in the CGL group vs 32.1% in the ABC one), and pts experiencing a PR were equally distributed between the two groups. Our data do not support the use of TMA to predict outcome in DLBCL. However, previously published data supporting the prognostic impact of TMA, have not enrolled pts with lymphomas of primary extranodal origin which, instead, were the majority in our study. Indeed, the study by Colomo et al, which enrolled 39% of pts affected by extranodal lymphomas, also failed to demonstrate a role of TMA in predicting outcome in DLBCL pts. Furthermore, the use of a limited number of antigens to define different subtypes of DLBCL may not be sufficient to identify the same patterns as defined by cDNA microarray technology.

Description: RATIONALE OF STUDY: Despite significant advances in prevention and therapy, cytomegalovirus (CMV) infection still represents an important cause of morbidity and mortality in patients undergoing allogeneic haematopoietic stem cell transplant (HSCT). The standard pre-emptive treatment is based on intravenous administration of Ganciclovir (GCV). Valganciclovir (VGC), the pro-drug formulation of GCV is characterised by an excellent bio availability, making this drug suitable for oral administration. PATIENTS: Since March 2003 all patients treated with reduced (27 patients) or fully ablative (3 patients) conditioning regimens followed by sibling HSCT, were monitored with bi-weekly CMV/PCR and pp65/assays. Overall 15 episodes of CMV positivity were detected in seven patients. Patients resulted positive (3 cells pp65+ or 1000/100000 PCR +) started oral treatment with VGC 900 mg bid, for the first fourteen days, followed by 900 mg q.d. up to at least seven days after assays normalization. The median duration of therapy was 21 days (range 10–21 days). No significant toxicity was observed. All patients had a normalization of CMV/PCR and pp65/assays within fourteen days, with a response rate (RR) of 100%. In two patients the oral VGC therapy was changed to the intravenous administration of Foscavir, because of concomitant neutropenia and acute GvHD. CONCLUSION: Pre-emptive treatment of CMV infection with VGC is safe, feasible and effective. Furthermore, the oral administration of this drug in an outpatient setting, reduces significantly the costs compared with a therapy that needs hospitalization as intravenous Ganciclovir.

Description: Haploidentical bone marrow transplantation using non-myeloablative conditioning and high-dose, post-transplant cyclophosphamide (Cy) has been shown to be a feasible approach for patients lacking an HLA identical donor with acceptable rates of acute and chronic Graft-versus-Host-Disease (GVHD); in the attempt to reduce the associated risk of graft failure and relapse incidence we decided to apply the same conditioning regimen using unmanipulated mobilized peripheral blood stem cells (PBSC). From June 2010 to December 2014, 31 consecutive patients affected by high-risk hematological malignancies, median age 51 years (range 19-70) (3 Acute Myeloid Leukemia, 6 Acute Lymphoblastic Leukemia, 9 Non-Hodgkin Lymphoma, 5 Multiple Myeloma, 2 Myelodysplastic Syndrome, 6 Hodgkin Lymphoma), with no available HLA identical donor (neither related or unrelated) underwent peripheral stem cell transplant from a haploidentical family donor. Disease status at transplant was the following: 13 complete remission (5 patients in 1°CR, 6 patients in 2°CR, 2 patients in 3°CR), 9 partial response (PR), 1 stable disease (SD), 7 progressive disease (PD), and a patient with myelodysplastic syndrome who received the transplant as upfront therapy. Conditioning regimen consisted of cyclophosphamide, fludarabine and TBI followed by infusion of haploidentical PBSC; post-transplant immunosuppression consisted of high-dose Cy on days +3 and +4, tacrolimus and micofenolate mofetile from day +5. A median of 5.7x106 (range 3.8-13.7) CD34+ cells/kg was infused with a median of 2.8x108 (range 0.3-5.4) CD3+ cells/kg. Patients readily engrafted with a median time to absolute neutrophil count ≥500/µL of 17.5 days (range 14-28) in 28/31 patients and a median time to platelet ≥20.000/µL of 21 days (range 11-60) in 27/31 evaluable patients. Three patients died of infection before engraftment, another patient showed myeloid engraftment but never recovered platelet counts and prematurely died of cytomegalovirus (CMV) disease. At a median follow-up of 366 days (16-1682), 16 patients are alive (55%): 13 patients are in CR, the other 3, all affected by Multiple Myeloma, have progressed and are undergoing other treatments, with a cumulative incidence of relapse of 33%. Fifteen patients have died; causes of death included progressive disease in 9 patients (60%) and infections in the remaining 6, with a cumulative transplant related mortality of 18%. CMV reactivation occurred in 15 of 27 patients at risk, at a median time of 34 days (range 22-55) after transplant, resolving with preemptive therapy in 14 patients and causing a fatal infection in 1 heavily pretreated patient. Grade I-II acute GvHD (aGvHD) occurred in 6/28 evaluable patients, with 1-year cumulative incidence of grade I-II aGvHD of 21% and no incidence of grade III-IV GvHD. Mild chronic GvHD (cGvHD) was observed in 3/27 evaluable patients with 1-year cumulative incidence of cGvHD of 11%. Achievement of mixed donor chimerism was rapid: 22/28 evaluable patients showed a CD3+ chimerism 〉50% by day +28. At day + 84, in 22/26 evaluable patients CD3+ cells chimerism was 〉90%, while 4/26 showed still a mixed donor chimerism: withdrawal of immunosuppression increased CD3+ chimerism in one patient to 90% at day +360; one patient developed hemolytic anemia and he is under immunosuppression, still a mixed chimera at day +401 while in CR; the other two died from infection at +251 and +361 never reaching the full donor chimerism. No graft failure was observed. Our data show that haploidentical non-myeloablative peripheral blood hematopoietic cell transplantation with high-dose post-transplant Cy is a feasible for patients lacking an HLA identical donor. The use of unmanipulated PBSC with the infusion of a greater number of CD3+ cells allows a rapid and sustained engraftment, reduces the risk of graft failure, does not appear to increase the risk of GVHD with a low/moderate relapse risk. Although the incidence of major infection was low, CMV reactivation remains a critical issues and further studies are needed to clarify recovery of CMV immunity and to reduce the overall treatment related toxicity. Disclosures No relevant conflicts of interest to declare.

Description: The aim of this prospective multicenter trial was to evaluate the efficacy of the nucleoside analogue 2-CdA in combination with Rituximab and to identify possible genetic factors that could influence the clinical response of the combination. From December 2003 to November 2006, 29 pretreated/newly diagnosed pts affected by WM were enrolled in the study. Pts characteristics included: sex (M/F) 9/19, median age 64 (range 36–75 yrs), a median IgM level before treatment of 2567mg/dL; 16 pts were newly diagnosed. The combination therapy was Rituximab at standard schedule (375 mg/mq) on day 1 followed by 2-CdA 0.1 mg/kg (sc injection) for 5 consecutive days. Each cycle was administered monthly for a total of 4 cycles. Clinical responses were evaluated two months after the end of treatment, according to Response Criteria from the 3rd International Workshop on WM. Expression analysis of the genes involved in the activity of 2-CdA (deoxycytidine kinase, deoxyguanosine kinase, 5′-nucleotidase, ribonucleotide reductase 1 and 2, human equilibrative nucleotide transporter and hCNT1) was performed on bone marrow cells, at baseline in 22 pts. The therapy was well tolerated, except in three patients who discontinued Rituximab due to cardiac toxicity during the first or second infusion. The treatment was delayed in 3 pts because of haematologic toxicity (G3 neutropenia) and in 2 of them the 2-CdA dose was reduced after 2 cycles. The only non-haematological complications observed were late respiratory infections which occurred in 3 pts. At a median follow-up of 21 (13–41) months we observed 17 (59%) CR/PR, 7 (24%) MR, 1 (3%) SD and 4 (14%) PD/NR. Pharmacogenomic analysis showed a statistically significant lower expression of the hCNT1 gene in pts who failed the treatment and who achieved a MR or SD than in those who achieved a CR or a PR (p=0.014) suggesting a possible relationship between reduced hCNT1 levels and a diminished clinical activity of 2-CdA. No significant difference was detected in the expression of the other genes analyzed. Our results suggest that the combination of 2-CDA and Rituximab is safe and active in WM pts; the use of pharmacogenomic analysis might contribute to a better selection of the patients who are more likely to respond to such a combination treatment.

Description: Abstract 5030 Treatment for multiple myeloma is dramatically changed over the past 5 years. Thalidomide containing regimen is now widely accepted as standard treatment for multiple myeloma in first line. The mechanism of action and the toxicity profile of Thalidomide, make this drug suitable for combination with chemotherapeutic agents. We report clinical results in terms of efficacy and safety of an antracycline containing regimen [liposomal doxorubicin (Myocet), Dexamethasone and Thalidomide (ThalDoDex)] In multiple myeloma before autologous transplantation. From June 2007 to June 2010, ThalDoDex was delivered to 28 previously untreated multiple myeloma patients. Median age was 59 years (range 42–71); 5 patients were staged IIA and 21 patients staged IIIA and 2 IIIB; 15, 8 and 5 patients were ISS I, II, III respectively. Fifteen patients presented IgG monoclonal immunoglobuline, 6 IgA, 5 patients light chains myeloma, 1 patient plasma cell leukaemia and one other secretory multiple myeloma. Treatment schedule was as follows: Thalidomide 100 mg/day for 14 days then 200mg/day until the end of induction; Dexamethasone 40 mg days 1à4; Liposomal Antracycline (MYOCET) 50 mg/sqm day 1 for 4 cycles at 4 weekly intervals. LMWH 100UI/kg/day was added to all patients for DTV prophylaxis. All patients were considered for a peripheral blood stem cell transplantation program according to age and clinical outcome. Twenty-five patients are evaluable for response. Fifteen patients (60%) achieved a CR (1 pt) or a nCR (5 pts), or a VGPR (9 pts) with an overall response rate of 80% (CR, nCR, VGPR, PR). Six patients developed transient febrile neutropenia which solved with antibiotics. During neutropenia two patients developed pneumonia treated with appropriate antimicrobial therapy. No major haematological toxicity was observed. No neurological toxicity or thrombotic event was reported. Our experience suggests that ThalDoDex is effective and safe as induction phase in newly diagnosed multiple myeloma patients. The clinical results are similar to those reported with other Thalidomide containing regimens. Liposomal doxorubicin in combination with Thalidomide and steroid may induce an important rate of CR and nCR as requested for the best clinical result of subsequent autologous transplant procedure. Our combination regimen avoiding alkylating agents and proteosome inhibitors may deserve the use at eventually subsequent relapse. Disclosures: No relevant conflicts of interest to declare.

Description: Haploidentical bone marrow transplantation using non-myeloablative conditioning and high-dose, post-transplant cyclophosphamide (Cy) has been shown to be a feasible approach for patients lacking an HLA identical donor with acceptable rates of acute and chronic Graft-versus-Host-Disease (GvHD); in the attempt to reduce the associated risk of graft failure and relapse incidence we decided to apply the same conditioning regimen using unmanipulated G-CSF mobilized peripheral blood stem cells (PBSC). From July 2010 to January 2014, 20 patients affected by high-risk hematological malignancies, median age 56 years (range 19-70) (2 Acute Myeloid Leukemia, 5 Acute Lymphoblastic Leukemia, 5 Non-Hodgkin Lymphoma, 3 Multiple Myeloma, 2 Myelodysplastic Syndrome, 3 Hodgkin Lymphoma), with no available HLA identical donor (neither related or unrelated) were enrolled in a prospective clinical protocol at our institution. Disease status at transplant was the following: 10 complete remission (6 patients in 1°CR, 3 patients in 2°CR, 1 patient in 3°CR), 4 partial response (PR), 3 stable disease (SD), 2 progressive disease (PD), and a patient with myelodysplastic syndrome who received the transplant as upfront therapy. Conditioning regimen consisted of cyclophosphamide, fludarabine and TBI followed by infusion of haploidentical unmunipulated mobilized PBSC; post-transplant immunosuppression consisted of high-dose Cy on days +3 and +4, tacrolimus and micofenolate mofetile from day +5. A median of 5.5x106 (range 3.8-13.7) CD34+ cells/kg was infused with a median of 2.8x108(range 0.3-5.4) CD3+ cells/kg in 19/20 evaluable patient: 1 patient was unable to receive the complete conditioning regimen due to rapid worsening of her clinical condition and therefore she was taken out of the study protocol. Patients readily engrafted with a median time to absolute neutrophil count ≥500/µL of 17.5 days (range 14-28) in 17/19 patients and a median time to platelet ≥20,000/µL of 21 days (range 11-53) in 18/19 evaluable patients. One patient died of infection and progressive disease before engraftment, another patient showed myeloid engraftment but never recovered platelet counts and prematurely died of cytomegalovirus (CMV) disease. At a median follow-up of 486 days (12-1297), median OS has not been reached with 13 patients being alive, 8 in CR, 3 in PR and 2 in PD. Causes of death included progressive disease in 3 patients and infections in the remaining 3, with a cumulative transplant related mortality of 15%. CMV reactivation occurred in 12 of 16 patients at risk, at a median time of 34 days (range 22-55) after transplant, resolving with preemptive therapy in 11 patients and causing a fatal infection in 1 heavily pretreated patient. Acute GvHD (aGvHD) occurred in 1 patient, with 1-year cumulative incidence of grade I/II aGvHD of 5.6% (95% CI:8.0-33.4%) and no incidence of grade III/IV GvHD. Mild chronic GvHD (cGvHD) was observed in 2 patients with 1-year cumulative incidence of cGvHD of 8.3% (95% CI:1.2-46.1%). Achievement of mixed donor chimerism was rapid: 16/18 evaluable patients showed a CD3+ chimerism 〉50% by day +28. At day + 84, in 14/16 evaluable patients CD3+ cells chimerism was 〉50%, in 6 of them it was 〉95%. Modulation and withdrawal of immunosuppression increased donor CD3+ chimerism in both of the two remaining patients to 67% and 74% at day +360 and +180, respectively. Notably, no graft failure was observed. Our data show that non-myeloablative haploidentical peripheral blood stem cell transplantation with high-dose post-transplant Cy is a feasible and safe approach for patients lacking an HLA identical donor. The use of unmanipulated, G-CSF mobilized PBSC with the infusion of a greater number of CD3+ cells allows a rapid and sustained engraftment, reduces the risk of graft failure and does not appear to increase the risk of GvHD. Although the incidence of major infection was low, CMV reactivation remains a critical issues and further studies are needed to clarify recovery of CMV immunity and to establish better prophylaxis therapy. Disclosures No relevant conflicts of interest to declare.

Description: Following an in vivo T cell depleting non-myeloablative conditioning regimen, 5 patients, aged 22–49, received combined kidney and bone marrow transplantation from a haploidentical related donor. Rituximab was included in the conditioning for patients 4 and 5. All patients developed initial mixed chimerism but lost it by day 21; no patient developed GVHD. Four patients discontinued immunosuppression from 240 to 422 days after BMT and have remained off immunosuppression for 9 to 52 months with no evidence of allograft rejection. Flow cytometry was used to assess lymphocyte subsets recovering after transplant. CD3 counts recovered slowly, exceeding 500 cells/μl at days +271, +365, +640 and +450. While memory CD45RO+ cells were most prevalent among CD4+ cells, naïve-type CD4+CD45RA+ cells, presumably arising from the recipient thymus, ranged from 8% to 56% at the time when total CD4 counts recovered to 〉100 cells/μl (days +165, +21, +352, +240). Notably, a very high proportion of initially recovering T cells were CD3+CD4+ expressing CD25 in all patients as early as day 7 and persisted over 1 year in 2 patients. At approximately day +120 and +365, we further characterized these cells for CD127, FOXP3, CD45RO, CD45RA, HLA-DR and CD62L expression. At Day +120, all 4 patients showed increased frequencies (10.7±4.6%) of CD25+CD127-FOXP3+ regulatory T cells (Treg) within the CD4 population compared to healthy subjects (3.8±0.4%). Expression of CD45RO, CD45RA, CD62L and HLA-DR was variable. By 1 year post-transplant, frequencies of Treg had decreased to levels similar to those in normal subjects. In vitro assays for CD8 and CD4 T cell-mediated alloreactivity (CML/MLR) showed development of long-lasting donor-specific unresponsiveness by 3 months after transplant in Patients 2, 4 and 5, and by 9 months in Patient 1. Responses to 3rd party recovered in all patients after a period of unresponsiveness. In Patient 1, in whom anti-donor CML reactivity declined gradually to become unresponsive by 9 months, depletion of CD4+CD25+ cells revealed a residual anti-donor CML and MLR response at 1year but not at 18 months. In 2 other patients, depletion of CD4+CD25+ cells did not reveal an anti-donor response at time points analyzed from day +122 to 2 years. In patients in whom renal tubular epithelial cells (RTEC) were cultured from the donor kidney, loss of killing activity against donor RTEC was observed post-transplant. The high percentage of Treg recovering early after transplant suggests that they may play a role in initial tolerance induction. This regulatory mechanism may be followed by later deletion of donor-reactive T cells. The variable ability to detect regulation of anti-donor reactivity may reflect the strength of the initial response, as patients with weak pre-transplant anti-donor responses and rapid post-transplant development of donor unresponsiveness did not reveal anti-donor response when Treg were depleted. In addition, infiltration of Treg at the graft site, not revealed by the assays described, might be responsible for tolerance in these patients.