Donor Cell Infusion Research Articles

Evaluation of 243 Patients with Myelodysplastic Syndrome (MDS) Referred for Hematopoietic Cell Transplant (HCT) Including Donor Availability and HCT Outcomes.

Abstract 3383Poster Board III-271Allogeneic hematopoietic cell transplantation (HCT) remains the only curative treatment strategy for patients (pts) with MDS. Until recently, the toxicity of HCT excluded older pts such as those with MDS from HCT. In July 2004 the Moffitt Cancer Center (MCC) implemented a pharmacokinetically targeted IV busulfan plus fludarabine (tBuFlu) induction regimen that was myeloablative, with reduced treatment related toxicity, permitting the expansion of empiric age limitations for HCT up to 75 years (yrs). From that time until August 2009, 243 new pts (NP) with a diagnosis of MDS or CMML were evaluated for HCT. An allogeneic donor was identified in 170 pts and 112 received an HCT at MCC, the majority using tBuFlu. (HCT) The median age of the 112 transplant pts at Moffitt was 55.6 yrs (24.8 – 73.5). Sixty pts were older than 55 yrs and 11 were above 65 years of age. All pts that proceeded to transplant had adverse risk factors including progression to AML (25), secondary MDS (30) or progression of disease. At the NP visit, IPSS risk was Low (11), Int-1 (35), Int-2 (42), and High (21) or not evaluable (3) (NE). Donors included 49 sibling donors (SIB), 48 matched unrelated donors (MUD) and 15 mismatched unrelated donors (mMUD). Median time from the NP visit to donor cell infusion was 197 days (98-1810) for mMUD; 132 days (64 – 488) for MUD and 119 days (25 – 613) for SIB. Median follow-up is 19.3 months (0 – 61.4) and the Kaplan-Meier overall survival (OS) from NP visit is 77 % at one year and 56 % at two years. Overall survival is not statistically different between SIB, MUD or mMUD HCT (P=0.39). In 58 pts who did not receive a HCT, a donor was identified. (Yes donor, No HCT) Median age was 57 yrs (19 – 69) with 28 pts older than 55yrs and 9 pts older than 65 yrs. At the NP visit, IPSS was Low (5), Int-1 (19), Int-2 (17), High (16) and NE (1). Ten had treatment related disease and 26 had progressed to AML. Donors included SIB (14), MUD (27), mMUD (15), and umbilical cord blood (2). Median follow up is 10.3 months (1.7 – 66.3). Reasons for not proceeding to HCT following the donor search included waiting for progression due to low risk disease or good response to therapy (9), progression of disease or death with ongoing donor search (21), new ineligibility for HCT (12), pt declined HCT (8), HCT elsewhere (3) and 5 pts are in active evaluation to proceed to HCT. Seventy-three pts did not obtain a donor (No Donor, No HCT). Median age was 64.4 yrs (32.6 – 72.1) with 65 pts older than 55yrs and 31 older than 65 yrs. IPSS at NP visit was Low (8), Int-1 (16), Int-2 (25), High (16) and NE (8). Twenty had treatment related disease and 18 had progressed to AML. The reasons for not proceeding to transplant were as follows; ineligibility for HCT or progressed/died during the donor search (27), insurance denial (15), patient declined HCT (10), low risk disease (5), no suitable donor found (9), and 7 are in active searches. Median follow up of these pts is 6.7 months (0.9 – 61.3). For the entire cohort (243 pts), pts with a donor had significantly improved overall survival vs pts with no donor (P=0.003); there was no difference in OS between pts with a donor who did not proceed to HCT and everyone without a donor (P=0.27). Twenty-eight of the 73 pts without a donor did not have donor searches due to ineligibility for HCT at NP visit, pt declined at NP visit or insurance declined even though the pt was eligible. If these are excluded, in the remaining 215pts OS for pts with an identified donor was improved compared to OS of those without a donor (P=0.009) and pts with a donor and no HCT had similar OS to those without an identified donor (P=0.3). These data demonstrate that 70% of patients can acquire a suitable allogeneic donor and 47% of patients seen as a HCT new patient consult can proceed to HCT for MDS with a significant overall survival advantage using a SIB, MUD or mMUD. Disclosures:No relevant conflicts of interest to declare.

Outcome of Relapse after Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) - The Effect of the Underlying Disease on Survival Following Relapse.

We have reported that the outcome of reduced-intensity allogeneic HSCT is not affected by the underlying diagnosis (Mehta et al, ASH 2006). With an additional year’s follow-up and a minimum follow-up of 18 months, this observation continues to remain valid in a series of 91 uniformly-treated patients with hematologic malignancies (19–71 years; median 53; 22% >60 years). 29 patients had acute leukemia, 38 had lymphoma (Hodgkin/NHL) or CLL, and 24 had myeloma. Two-thirds of the patients had refractory disease. Performance status (PS) was 0 in 31%, 1 in 47%, and 2/3 in 22%. Donors (55% aged >45 years) were HLA-matched siblings (n=51), 10/10 allele-matched unrelated individuals (n=29), or 9/10 allele-matched related/unrelated individuals (n=11). The conditioning regimen comprised 100 mg/m2 melphalan with (if no prior autograft) or without (prior autograft) 50 mg/kg cyclophosphamide. GVHD prophylaxis comprised cyclosporine-mycophenolate (HLA-matched sibling donors) or tacrolimus-mycophenolate (other type of donors). G-CSF or GM-CSF were not given routinely to accelerate engraftment. Supportive care was uniform. As the figure shows, the underlying disease did not affect overall survival (OS) after HSCT. Figure Figure The underlying disease did not affect disease-free survival, relapse risk or transplant-related mortality. 51 of these patients relapsed. Relapsed disease was treated with immunotherapy (withdrawal of immunosuppression and/or donor cell infusion), and/or non-immune salvage treatment options. The latter included conventional chemotherapy for all 3 disease groups, monoclonal antibodies for NHL, and drugs such as thalidomide, bortezomib and lenalidomide for myeloma. Some patients with poor performance status received only supportive therapy. 43 of the 51 relapsing patients, including all with acute leukemia, died of progressive disease or complications of salvage therapy. The 8 survivors have lymphoma or myeloma, and are in remission or have stable disease. As the figure shows, OS after relapse was significantly affected by the underlying disease. Figure Figure Other factors affecting OS significantly after relapse were LDH and performance status. These data suggest that the outcome of patients with hematologic malignancies is generally poor if the disease relapses after reduced-intensity HSCT. However, some patients with lymphoma or myeloma can survive for prolonged periods after relapse because of effective salvage therapy options. Patients with acute leukemia fare poorly because of lack of effective salvage therapy, and exploration of strategies such as a second allograft may be appropriate for them.

Improved Outcome From Invasive Adenovirus Infection in Pediatric Patients After Hemopoietic Stem Cell Transplantation Using Intensive Clinical Surveillance and Early Intervention

Adenovirus is a common cause of morbidity and mortality after hemopoietic stem cell transplantation in children. Recently the incidence, risk factors, and outcome of such infections have been better defined using improved virologic detection methods, in particular polymerase chain reaction. We have introduced intensive virologic surveillance for adenovirus in our institution including at least weekly polymerase chain reaction testing of blood and stool samples. We report on 71 prospectively monitored transplants, including 40 from unrelated donors. In total, there were 8 cases of invasive adenovirus infection, 3 of whom died. Mortality was less than in previous studies as cases were managed with antiviral chemotherapy and reduction of immune suppression. In fatal cases, there was concurrent difficult graft versus host disease making withdrawal of immune suppression therapy impossible. We describe 2 cases of graft failure in association with adenovirus viremia and its treatment that were successfully managed with further donor cell infusion.

Delayed Onset GVHD Following Reduced Intensity Conditioning HSCT Is Associated with Persisting Host DC and Late Emergence of Activated Donor DC.

Allogeneic haematopoietic stem cell transplantation (HSCT) has relied on myeloablative conditioning (MAC) to ablate malignancy and control graft rejection. Conditioning is vital for HSC engraftment, however it up-regulates inflammatory cytokine and endotoxin levels, activates dendritic cells (DC) and initiates GVHD. Non-myeloablative, but immunosuppressive, reduced intensity conditioning (RIC) is increasingly used to combat treatment related mortality (TRM) associated with MAC. RIC reduces TRM and delays GVHD onset, but overall GVHD incidence is unchanged. We hypothesise that this is due to persistence of host DC.No clinically relevant in vivo murine models of RIC allogeneic HSCT have been reported to date, preventing studies of the mechanisms of RIC-associated delayed-onset GVHD. We developed two murine models of pre-transplant conditioning, which result in GVHD after infusion of donor cells [either MHC mismatched (UBI-GFP/BL6→BALB/c) or MHC-matched minor antigen mismatch (miHA; UBI-GFP/BL6→BALB.B), the latter mimicking allogeneic HLA-identical sibling HSCT]. These models combine clinical doses of cytotoxics (cyclophosphamide and fludarabine) with radiation, enabling us to monitor effects of these regimens on DC subsets and activation daily during conditioning and thereafter. GVHD onset is significantly delayed after RIC compared to MAC in HSCT recipients in MHC mismatched (MAC d+6 vs. RIC d+18–35; p<0.001) and miHA mismatched (MAC d+18–35 vs. RIC d+44–90; p<0.001) HSCT models (Fig 1). In HSCT experiments mice were sacrificed at defined time points post-HSCT and also when severe GVHD required sacrifice. In MHC mismatched RIC HSCT recipients, mice with severe GVHD had higher levels of donor engraftment in BM and spleen than those treated with MAC (Fig 2A). Timed sacrifice data showed persistence of activated (CD11c+MHCII+) host DC in BM of RIC treated mice until D+21, but this was tempered by an increase in activated donor DC (Fig 2B). This was in direct contrast to MAC treated mice, which maintained low levels of activated host DC (Fig 2B). In addition, activated DC present in BM of RIC HSCT mice at death due to GVHD are primarily of donor origin as compared with MAC HSCT mice (Fig 2C). Delayed emergence of activated donor DC in RIC treated mice preceded delayed onset GVHD in this model.These findings suggest that the major factors contributing to delayed-onset GVHD post-HSCT in RIC recipients are persistence of host DC and delayed emergence of activated donor DC. Delineating the effect of MAC and RIC regimens on DC provides a rationale to target activated DC as GVHD therapy. [Display omitted] [Display omitted]

Induction of Anti-Cancer Effects by Targeting Alloreactive Intentionally Mismatched T Cells and NK Cells to Tumor Cells Using Bi-Specific Trifunctional Antibodies.

Engraftment of donor stem cells induces host vs graft unresponsiveness thus allowing durable engraftment of donor lymphocytes which can induce graft vs malignancy (GVM) effects following transplantation of MHC compatible donor stem cells. Unfortunately, graft vs host disease (GVHD) frequently results in major toxicity and mortality and limits the clinical efficacy of cell therapy. Although the intensity of GVM effects can be improved by using mismatched donors GVM effects cannot be induced in recipients of haploidentically mismatched allografts because of anticipated lethal GVHD. We have attempted to induce GVM effects by intentionally mismatched donor lymphocytes by targeting donor derived T cells and NK cells activated by rIL-2 to cancer cells, in order to improve the efficacy of the GVL effects on the one hand and prevent or minimize GVHD by targeting alloreactive cells to the tumor cells that need to be eliminated using bispecific tri-functional antibodies, on the other. A trifunctional bispecific antibody (BiLu) directed against murine CD3 and human epithelial-cell adhesion molecule (EpCAM), was administered concomitantly with naïve or IL-2 activated donor splenocytes to mice inoculated with a murine model of melanoma cells transfected with human EpCAM (B16-EpCAM). A total of 10/20 and 32/38 mice treated with BiLu and naïve or IL-2 activated donor splenocytes, respectively, were tumor-free survivors without GVHD for >250 days following tumor inoculation. Out of 28 of the disease free survivors (DFS) without GVHD, treated with IL-2 activated splenocytes and BiLu, 24 mice were resistant to a second tumor inoculum (104) given >250 days following the first tumor inoculation. In contrast, only 8 of the 18 DFS mice treated with naïve splenocytes and BiLu, and 5 of the 10 DFS controls treated with BiLu only, resisted the second tumor challenge. Induction of anti-tumor immunity by IL-2 activated cells and BiLu was more efficient and long-lasting in mice previously injected with a lethal first tumor cell dose (5×104) than in mice inoculated with a sub-lethal tumor cell dose (5×103). Interestingly, a similar infusion of donor cells labeled with bi-specific anti-EpCAM and anti-CD3 following allogeneic stem cell transplantation for metastatic breast cancer did not result in augmentation of GVHD. In conclusion, concomitant inoculation of alloreactive donor lymphocytes and trifunctional bispecific antibody can be effective for targeting of killer T and NK cells to tumor cells while avoiding GVHD, as well as for induction of long-lasting anti-cancer immunity, most likely due to presentation of tumor antigen to T cells by antigen presenting cells (dendritic cells) expressing Fc receptor binding to the Fc portion of the bi-specific antibody.

Monitoring Antidonor Alloantibodies as a Predictive Assay for Renal Allograft Tolerance/Long-term Observations in Nonhuman Primates

In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients. Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4). All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR. ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.

G-CSF–treated granulocytes inhibit acute graft-versus-host disease

It has been shown that in vivo and in vitro treatment with G-CSF induces the generation of low-density granulocytes (LDGs), which copurify with PBMCs and inhibit IFN-gamma production by human T cells. These results prompted us to postulate an immunomodulatory role for LDGs in acute graft-versus-host disease (aGVHD). Here it is shown that in the mouse experimental model, in vivo and in vitro G-CSF treatment generates LDGs capable of inhibiting 80% of T-cell IFN-gamma production. To assess the role of these LDGs in aGVHD, lethally irradiated (C57BL/6 x BALB/c) F1 hosts were reconstituted with T cell-depleted bone marrow cells plus nylon wool-purified spleen cells from G-CSF-treated (G-NWS) or -nontreated (NWS) C57BL/6 donors. Recipients of G-NWS had a 75% survival rate in contrast to a rate of 25% in the NWS recipients. The protective effect was completely abolished, and the mortality rate was 100% if donor-cell infusion was treated with anti-Gr1. Moreover, if LDGs were infused with NWS, full protection of aGVHD was observed, and no signs of disease were evidenced by mortality rate, weight loss, or histopathology of target organs. These results revealed the unexpected immunosuppressive capacity of G-CSF based on the generation of LDGs, leading to the possibility of using these cells as inhibitors of aGVHD.

Haploidentical Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation (HCT) Using Total Lymphoid Irradiation (TLI) and Anti-Thymocyte Globulin (ATG) Conditioning Protects Against Acute Graft Versus Host Disease (GVHD).

Murine models of transplantation established that nonmyeloablative conditioning using repeated low doses of irradiation targeted to lymphoid tissues (TLI) and depletive anti-T cell antibodies protects against GVHD by skewing residual host T cell subsets to favor regulatory natural killer (NK) T cells that suppress GVHD by polarizing donor T cells toward secretion of non-inflammatory cytokines such as IL-4. We recently translated the murine protocol to a clinical study using non-myeloablative TLI and ATG host conditioning with HLA matched related and unrelated donors, and showed a marked reduction in the incidence of acute GVHD while retaining graft anti-tumor activity (Lowsky et al., in Press NEJM). Engrafted donor CD4+ T cells showed a marked increase in IL-4 production as compared to CD4+ T cells from controls. We now adapted the TLI and ATG nonmyeloablative host conditioning regimen to a clinical study of allogeneic HCT using haploidentical matched (3/6 HLA matched) related donors to determine if it will result in donor hematopoietic cell engraftment and also protect against acute GVHD. Blood derived hematopoietic progenitor cells were collected by apheresis from donors mobilized with G-CSF and the product was T cell depleted using CD34+ selection. CD3+ T cells were added back to the donor inoculum according to a dose escalation schedule. The initial T cell dose was 1 x105 CD3+ cell/kg with designated increments based on clinical outcomes of up to a maximum of 1 x107 CD3+ cells/kg. The desired CD34+ cell dose was >5 x 106 CD34+ cells/kg for all patients. Seven patients were transplanted; the median age was 53 years (range 27 to 61 years). Five patients had acute myelogenous leukemia, two with disease in remission and three not in remission at the start of TLI and ATG, one with myelodysplastic syndrome, and one with progressive peripheral T cell lymphoma. The median follow-up for all patients is 265 days with three of seven patients alive and free of disease at the last observation period. Sustained donor hematopoietic cell engraftment was achieved in three of three patients only after the T cell dose was increased to 1 x107 CD3+ cells/kg. No patient developed acute GVHD. None of the three patients receiving the highest dose of T cells had any invasive fungal or viral infections. Monitoring of sorted host T cell subsets before TLI and ATG, and immediately after but before the infusion of donor cells, revealed in five of five patients a highly significant skewing of residual host T cells favoring invariant NK T (CD3+ CD161hi Va24 +Vb11 +) cells. The mean absolute number of host CD3+, and CD4+ and CD8+ T cells decreased by 99, 163 and 121 fold, respectively, immediately after conditioning compared to the absolute numbers before the start of TLI and ATG, whereas the mean absolute number of invariant NK T cells decreased by only 11%. In conclusion, we have determined the conditions for successful hematopoietic cell engraftment using a non-myeloablative regimen of TLI and ATG that appears associated with a reduced aGVHD risk yet retained graft anti-tumor activity. As in the pre-clinical model, we show direct evidence that the low incidence of aGVHD is associated with a significant alteration in residual host T cell subsets markedly favoring invariant NK T cells.

CD3 Depleted Hematopoietic Peripheral Blood Stem Cell Grafts in Children with Refractory Hematologic Malignancies Undergoing Transplantation from Mismatched Related Donors.

Allogeneic HSCT is the only curative intervention for patients with persistent disease or who recur after transplantation; however, these patients are often not considered for HSCT because of their persistent disease or high risk for regimen-related toxicity. We conducted a prospective study for patients who had hematologic malignancies with refractory disease or who relapsed after allogeneic HSCT using mismatched family member donors and a reduced intensity conditioning regimen in an effort to allow GVHD to occur to reduce disease recurrence in this high risk patient population. The conditioning regimen consisted of fludarabine (40 mg/m2/day for 5 days), melphalan (60 mg/m2/day for 2 days), and thiotepa (10 mg/kg/day for one day). One dose of melphalan was omitted in 6 patients who were aplastic at the time of transplantation. OKT3 was administered from day −9 to +17 for prevention of graft rejection. GVHD prophylaxis consisted of MMF initiated on day −2. Rituximab 375 mg/m2 was administered on day 0 as EBV prophylaxis. Patients received G-CSF starting on day +6 until ANC ≥ 2000/mm3 for two consecutive days. Peripheral blood grafts were obtained after mobilization with G-CSF and GM-CSF. Grafts were depleted of T-lymphocytes on the CliniMACS device using the anti-CD3 antibody OKT3. 25 patients were treated in this manner: 10 with refractory disease and 15 requiring another allogeneic HSCT (14 had one prior HSCT, one had 2 prior HSCT). For refractory patients, diagnoses included AML (2 secondary AML, 1 persistent disease (PD)) JMML (n=1, PD), ALL (n=3, PD), and NHL (n=3, PD including one after autologous HSCT). For patients who had failed prior allogeneic HSCT, diagnoses included AML (n=7), ALL (n=7), and CML (n=1, blast crisis). Patients had failed HSCT from matched sibling donors (n=5), unrelated donors (n=5), unrelated cord blood grafts (n=2), and haploidentical parents (n=3). Patients were a median of 11 years old at HSCT (range, 1–26). The median number of CD34+cells/kg infused was 13.64 x 106/kg (range, 2.23–42.46); the median number of CD3+ cells/kg infused was 0.122 x 106/kg (range, 0.006–0.45). Two patients suffered primary graft rejection: one with refractory JMML recovered with persistent disease after OKT3 and a re-infusion of paternal PBSCs. The second underwent infusion of the original unrelated donor cells and engrafted. The 23 evaluable patients had a median time to ANC ≥ 500/mm3 of 10 days (range, 7–12) post-HSCT. One patient undergoing second HSCT developed secondary graft rejection requiring infusion of original sibling donor marrow. 13 patients developed acute GVHD, but only 2 developed grade 3–4 acute GVHD. 5 patients developed chronic GVHD. None developed VOD. Of the refractory patients, 7 died of relapse and 1 of regimen-related toxicity. Of those undergoing subsequent HSCT, 6 died of relapse and 2 of regimen-related toxicity. With a median followup of 472 days, (range, 147–767), 9 remain alive. Transplantation of mismatched related donor PBSC grafts using OKT3 for ex vivo T-cell depletion following a reduced intensity conditioning regimen produces favorable outcomes with acceptable toxicity in this this high-risk patient population.

Lin−CD117+ Hematopoietic Cells Preferentially Home to Spleen and Their Migration Is Affected by Selectins.

In hematopoietic stem and progenitor cell transplantation, infused donor cells home to recipient bone marrow (BM) where they become residents and sustain normal hematopoiesis. Understanding the molecular mechanisms that regulate hematopoietic cell homing might help to increase homing efficiency and to optimize cell engraftment in patients. When BM cells from enhanced green fluorescence protein (GFP) transgenic mice were transplanted into normal C57BL/6 recipients, GFP+Lin− cell recovery in BM peaked to 17 ± 2 %, 21 ± 8 %, and 22 ± 14 % at 18, 48 and 72 hours after cell infusion; likewise, GFP+Lin− cell recovery in the spleen peaked to 7 ± 3 %, 10 ± 0 %, and 14 ± 3 % at these time points. Conversely, GFP+Lin− cells were not detected in recipient thymus at any time. Of the recovered GFP+Lin− cells in the BM and spleen, GFP+Lin−CD117+ cells preferentially homed to the spleen (7.0 ± 0.5 % recovery in spleen vs. 0.3 ± 0.5 % recovery in BM; P<0.0001) since only 0.5 ± 1.1 % of recovered GFP+Lin− cells in the BM were CD117+, compared to a 25 ± 1.1% CD117+ recovery rate in the spleen (P<0.0001). Total body irradiation did not increase but instead reduced GFP+Lin− cell homing in both BM (P<0.002) and spleen (P<0.005). Also, the proportion of CD117+ cells among recovered GFP+Lin− cells was much lower (P<0.05) in irradiated than in unirradiated recipients. When sorted GFP+Lin−CD117+ and GFP+Lin−CD117− cells were transplanted into sublethally-irradiated recipients at 20–180 ×103 cells per recipient, no GFP+ cells reached detectable level in either the spleen or BM. When the same number of GFP+Lin−CD117+ and GFP+Lin−CD117− cells were co-transplanted with 20 ×106 normal B6 BM cells, we detected 0.15% to 0.86% cell recovery in recipient BM and spleen, indicating that total number of injected cells affects cell homing and recovery. All recovered cells carried the same GFP+Lin−CD117+ or GFP+Lin−CD117− phenotype as they were injected, indicating that there was no phenotype switch during the first 18 hours of homing. In a short-term engraftment assay, 81 ×103 GFP+Lin−CD117+ cells transplanted along with 20 ×106 normal BM cells resulted in 1.7–3.8%, 3.2–5.2% and 11–17% engraftment in recipient spleen, BM and peripheral blood respectively at 10 days after transplantation, while 96 ×103 GFP+Lin−CD117− cells transplanted along with 20 ×106 normal BM cells produced in no detectable engraftment. Similarly, in a long-term engraftment assay, donor engraftment was detected from GFP+Lin−CD117+ but not from GFP+Lin−CD117− cells when recipients were monitored at 15, 30, 60 and 90 days after transplantation. To test the role of selectin proteins in hematopoietic cell homing, we transplanted 20 ×106 GFP+ BM cells to B6 recipients that carry targeted deletions for E-, L- and P-selectins. At 18 hours, GFP+Lin- cell recovery was at 5.0 ± 0.4 % and 9.8 ± 1.8 % in the spleen and 19 ± 1.4 % and 11 ± 1.9 % in the BM for normal and selectin-deficient recipients. In conclusion, GFP+Lin− cells homed to BM and spleen but not to thymus, indicating that an appropriate microenvironment is necessary for adequate homing. Irradiation did not attract Lin− cells to the thymus but reduced Lin− cell homing to BM and spleen. While only a very small fraction of GFP+Lin−CD117+ cells homed to BM, they are likely the cells responsible for short- and long-term hematopoietic engraftment in recipients. Deletion of selectin genes may alter the microenvironment and shift Lin- cell homing to spleen, thus reducing hematopoietic cell homing to BM.

Author reply

We are delighted that Drs. Jourdan and Dastugue of the Bordeau, Grenoble, Marseille, Toulouse (BGMT) intergroup confirmed most of our findings in de novo acute myeloid leukemia (AML) with t(6;11)(q27;q23). In light of our experience, it would be interesting to know whether any of the patients in the BGMT series underwent transplantation, including the patient who survived for 47 months. We are now able to provide additional follow-up information on the two long-term survivors from our article.1 At last follow-up, Patient 1 remained alive and in remission at 73 months after diagnosis. However, Patient 2, who was the only Cancer and Leukemia Group B (CALGB) patient to undergo allogeneic transplantation in first complete remission (CR1), developed a recurrence nearly 6 years after undergoing transplantation. Cytogenetic analysis at the time of recurrence revealed t(6;11)(q27;q23) and del(13)(q12q21) in all 15 metaphase cells studied. The patient achieved a second CR after reinduction chemotherapy followed by infusion of additional donor cells and remained in remission at last follow-up 5 months later, 76 months after initial diagnosis. In addition, we recently identified another patient with de novo AML and t(6;11) from CALGB 9621. The patient was an African-American man age 23 years who presented with AML (French–American–British subtype M1), white blood cell count 1 × 109/L, hemoglobin 10 g/dL, platelets 138 × 109/L, and no organ involvement. The karyotype at diagnosis was 46,XY,del(5)(q13q33), t(6;11)(q27;q23)[20]. He achieved a CR with induction therapy and underwent an autologous transplantation in CR1, but he developed a recurrence at 14 months and died at 24 months after diagnosis. These data are consistent with those reported previously on the CALGB and literature patients and further support the notion that t(6;11) occurs with greater frequency among African Americans.1 Clearly, additional basic research and new therapies are necessary for this rare but high-risk subset of patients. Elucidation of the role of the AF6 gene, the fusion partner of the MLL gene in t(6;11)(q27;q23), may help to identify new targets for therapy. For example, AF6 may have an important role in Ras-dependent activation of the Ras-Raf protein kinase cascade.2 If the interaction of AF6 and Ras is perturbed in t(6;11) AML, then targeting Ras activity through new agents, such as farnesyl transferase inhibitors, may be an attractive investigational therapy in this rare form of leukemia. William Blum M.D.*, Krzysztof Mrózek M.D., Ph.D.*, Amy S. Ruppert M.A.S.* , Clara D. Bloomfield M.D*, * For Cancer and Leukemia Group B, Division of Hematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, Cancer and Leukemia Group B Statistical Center, Durham, North Carolina.

Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates.

In utero hematopoietic stem cell transplantation could potentially be used to treat many genetic diseases but rarely has been successful except in severe immunodeficiency syndromes. We explored two ways to potentially increase chimerism in a nonhuman primate model: (a) fetal immune suppression at the time of transplantation and (b) postnatal donor stem cell infusion. Fetal Macaca nemestrina treated with a combination of the corticosteroid betamethasone (0.9 mg/kg) and rabbit thymoglobulin (ATG; 50 mg/kg) were given haploidentical, marrow-derived, CD34+ -enriched donor cells. Animals treated postnatally received either donor-derived T cell-depleted or CD34+ -enriched marrow cells. Chimerism was determined by traditional and real-time polymerase chain reaction from marrow, marrow progenitors, peripheral blood, and mature peripheral blood progeny. After birth, the level of chimerism in the progenitor population was higher in the immune-suppressed animals relative to controls (11.3% +/- 2.7% and 5.1% +/- 1.5%, respectively; p = .057). Chimerism remained significantly elevated in both marrow (p = .02) and fluorescence-activated cell sorted and purified CD34+ cells (p = .01) relative to control animals at > or = 14 months of age. Peripheral blood chimerism, both at birth and long term, was similar in immune-suppressed and control animals. In the animals receiving postnatal donor cell infusions, there was an initial increase in progenitor chimerism; however, at 6-month follow-up, the level of chimerism was unchanged from the preinfusion values. Although fetal immune suppression was associated with an increase in the level of progenitor and marrow chimerism, the total contribution to marrow and the levels of mature donor progeny in the peripheral blood remained low. The level of long-term chimerism also was not improved with postnatal donor cell infusion.

The impact of portal infusion with donor-derived bone marrow cells and intracellular cytokine expression of graft-infiltrating lymphocytes on the graft survival in rat small bowel transplant model

Background Intraportal administration of alloantigen is reported to reduce antigen-specific immune responses, although the underlying mechanisms for the reduced immunological reactions, especially those of the graft, are poorly understood. We examined intracellular cytokine production by graft-infiltrating lymphocytes (GILs) and peripheral blood lymphocytes (PBLs) to elucidate the underlying mechanisms of beneficial effects on intraportal infusion of donor cells in rat small bowel transplantation (SBT). Methods Recipient rats (Lewis) were transplanted with small bowel from ACI rats. Tacrolimus (Tac) was injected daily from days 0 to 4. Bone marrow cells of ACI rats were infused via the portal or tail vein on the day of surgery. On day 5, both GILs and PBLs collected from SBT graft and peripheral blood, respectively, were analyzed for intracellular cytokine production of recipient-derived αβ-T cells. The Th1/Th2 balance in each group was designated as the ratio of the percentage of GILs or PBLs staining positive for intracellular IL-4 or IFN-gamma, respectively. The total cell numbers of GILs from SBT graft were also counted. Results Survival of recipients was markedly prolonged by the combination of Tac and donor-specific bone marrow infusion via the portal vein (DS BMI-PV-Tac) compared with the untreated control, Tac alone, or DS BMI tail vein plus Tac. DS BMI-PV-Tac significantly decreased the total cell numbers of GILs and also induced remarkable Th2-type response in GILs. Conclusions Our results indicate that DS BMI-PV-Tac decreased GILs and enhanced Th2-type response in SBT graft, both of which are associated with a significant prolongation of graft survival in SBT.

LONG TERM OBSERVATIONS IN RENAL ALLOGRAFT RECIPIENTS TREATED WITH A CHIMERISM- INDUCING NONMYELOABLATIVE REGIMEN WITH CD154 BLOCKADE

O111* Aims: Cynomolgus monkey renal allograft recipients treated with a chimerism-inducing regimen with perioperative CD154 blockade were studied long-term (>5 years) to establish reliable tolerance assays that are capable of predicting the long-term outcome. Methods: Cynomolgus monkeys in Group A were treated with a conditioning regimen consisting of low dose total body irradiation (TBI 1.5 Gy x2), thymic irradiation (TI 7 Gy), anti-thymocyte globulin (ATG), perioperative anti-CD154 monoclonal antibody (mAb) treatment, combined donor bone marrow (DBM) and kidney transplants on day 0 and one month course of cyclosporine (CyA). No immunosuppression was given after day 28. Control Groups include Group B: donor splenocyte infusion instead of DBM, Group C: no donor cell infusion, or Group D: no TI. Results: All eight recipients in Group A developed multilineage chimerism up to day 100 and 7/8 survived long-term after discontinuation of immunosuppression (>1850, >1323, 837, 755, 401, 373, 206, 58 days). Four of seven long-term survivors did not developed any evidence of rejection with the longest survival exceeding five years. Two of four long-term recipients accepted donor skin graft indefinitely while rejecting third-party skin graft within 10 days. However, 3/8 recipients in Group A eventually developed chronic rejection. All recipients in control groups B and C did not develop chimerism and lost their kidney allograft function either by acute or chronic rejection (CR) (292, 274, 250, 113, 93, 51 days). 2/3 recipients in Group D developed chimerism and one of them survived long-term without rejection (289, 71, 44). Anti-donor alloantibody (ADA), measured by flow cytometry and C4d staining of the graft biopsy were significantly relevant to CR. All six recipients that developed ADA eventually developed CR. Allograft biopsies of all six recipients with positive ADA later developed C4d in their renal allograft biopsies. In contrast, five recipients without ADA have never developed C4d or CR. In Group A, there was no significant difference found between tolerant recipients and rejecters in the level and duration of mixed chimerism or postoperative MLR and CML. In the ELISPOT assay, where posttransplant peripheral blood mononuclear cells (PBMC) were activated via either direct (donor APC) or indirect allorecognition pathway (sonicated donor cells), tolerant recipients showed specific inhibition of donor-reactive, gamma-interferon (gIFN) production in the indirect pathway (6.5 ±6 spots per 106 PBMC). In contrast, the recipients with CR showed vigorous gIFN production (132 ±17). In the direct pathway, however, there was no significant difference detected in gIFN production between tolerant recipients and rejecters (31.5 ±15 vs 82 ±25). Conclusions: Long-term stable renal allograft survival was achieved in 50% of recipients treated with a chimerism inducing nonmyeloablative regimen with perioperative CD154 blockade. Posttransplant ADA by flow cytometry, positive C4d in renal biopsies and positive gIFN production in the indirect pathway appear to correlate with the development of chronic rejection.

Microchimerism does not correlate with survival of murine cardiac allografts

The development of microchimerism was evaluated at different time points after infusion of a mixed population of bone marrow and spleen cells from (BALB/c × C57Bl/6)F1 mice in the presence or absence of a cardiac transplant. Microchimerism was observed in the spleen, bone marrow and thymus of transplanted BALB/c mice even after graft rejection. In the absence of transplantation, donor cells persisted especially in the thymus. The results show that despite augmentation of graft survival after donor cell infusion compared to nontreated controls, the development of microchimerism did not sustain cardiac semihistocompatible grafts. Moreover, the persistence of donor cells in the thymus in both situations suggests a role for this organ in the increased graft survival in our model.

Donor-derived hematopoietic cells in organ transplantation: a major step toward allograft tolerance?

Infusion of donor-derived cells can improve organ allograft survival in animal models. Under certain conditions, it can even induce tolerance (i.e., unlimited organ survival without any maintenance immunosuppressive therapy). Use of nonmyeloablative regimens allows engraftment of donor-derived bone marrow cells, induction of mixed chimerism, and tolerance in rodents. High doses of bone marrow cells together with anti-T-cell antibodies can even result in mixed chimerism without cytoablative host conditioning. Cultured donor-derived CD34+ cells or donor-derived immature (or even mature) dendritic cells associated with monoclonal antibodies directed against co-stimulatory molecules might also induce tolerance. Among the numerous experimental protocols leading to tolerance of solid organs in animal models, how can we find our bearings in human transplantation? Numerous problems have yet to be solved: the type and amount of donor-derived cells (including stromal cells) to be used, the timing for infusion of donor cells in keeping with organ transplantation, the route of infusion (should it be intravenous, into the portal vein?), and the conditioning regimen. The first clinical trials would appear to indicate that tolerance induction in humans using donor-derived cells is a relatively safe solution that is both promising and realistic.

Promotion of skin graft tolerance across MHC barriers by mobilization of dendritic cells in donor hemopoietic cell infusions.

Flt3 ligand (FL) dramatically increases the number of immunostimulatory dendritic cells (DC) and their precursors in bone marrow (BM) and secondary lymphoid tissues. Herein we tested the ability of FL-mobilized donor hemopoietic cells to promote induction of skin graft tolerance across full MHC barriers. C57BL/10 (B10; H2(b), IE(-)) mice were given 10(8) spleen cells (SC) from normal or FL-treated, H-2-mismatched B10.D2 (H2(d), IE(+)) donors i.v. on day 0, 200 mg/kg i.p. cyclophosphamide on day 2, and 10(7) T cell-depleted BM cells from B10.D2 mice on day 3. B10.D2 skin grafting was performed on day 14. Indefinite allograft survival (100 days) was induced in recipients of FL-SC, but not in mice given normal SC. Tolerance was associated with blood macrochimerism and was confirmed by second-set skin grafting with donor skin 100 days after the first graft. In tolerant mice, peripheral donor-reactive T cells expressing TCR Vbeta11 were deleted selectively. Immunocompetence of tolerant FL-SC-treated mice was proven by rapid rejection of third-party skin grafts. To our knowledge this is the first report that mobilization of DC in donor cell infusions can be used to induce skin graft tolerance across MHC barriers, accompanied by specific deletion of donor-reactive T cells.

Delayed low-level calcineurin inhibition promotes allospecific tolerance induction by posttransplantation donor leukocyte infusion.

Donor lymphocytes infused after organ transplantation can have strong immunoregulatory effects. Application of such protocols for transplant tolerance induction in a clinical setting will, however, require combination of specific immunomodulatory strategies with nonspecific immunosuppressive medication for safety reasons. The aim of this study was to analyze the effects of immunosuppressive treatment on tolerance induction protocols by posttransplantation donor lymphocyte infusion. The interaction of postoperative donor leukocyte infusion with different types, dosage, and timing of immunosuppressive drugs were studied in a rat model of heart transplantation. Tolerance could be achieved if donor cell infusion was combined with delayed, but not immediate, low-dose cyclosporine treatment, and this was associated with activation and apoptosis of host lymphocytes. In contrast, combinations with an antibody against the interleukin 2 receptor led to long-term graft survival but severe chronic rejection, and combinations with high-dose cyclosporine or sirolimus led to acute rejection. Postoperative donor leukocyte infusion is a potential way for tolerance induction, but the type, dose, and timing of medication are highly critical for its efficacy.

An essential role for natural killer cells in augmentation of allograft survival mediated by donor spleen cells.

Previous studies have shown that skin allograft survival can be augmented by the administration of donor spleen or donor bone marrow in antithymocyte serum (ATS) treated recipients. Because natural killer cells (NK) have been reported to possess immunoregulatory properties, we investigated whether the ability of donor spleen or bone marrow cells to enhance allograft survival was dependent on the presence of donor NK cells. Recipient (C57BL/6 x A/J)F1 strain mice (H2 haplotypes Kb/k, Ab/k, E-/k, Db/d) were treated with ATS on days -1 and +2 relative transplantation of a C3H (H-2k) skin allograft. On day +7, each recipient was randomly assigned to one of the following groups that received i.v. donor C3H cell infusions via the tail vein: 1) 5.0x10(7) wild-type donor spleen cells (SPC); 2) 5.0x10(7) spleen cells from C3H/HeJ-Lystbg-2J/+ mice (commonly called beige mice and have selectively impaired NK cell function); 3) 2.5x10(7) wild-type donor bone marrow cells (BMC); 4) 2.5x10(7)beige C3H bone marrow cells; and 5) no donor cell infusion (ATS controls). In another experiment, each recipient was randomly assigned to one of the following groups that received injections of: 1) 4.75x10(7) spleen cells depleted of NK cells; 2) 2.5x10(6) purified splenic NK cells; 3) a coinfusion of 5.0x10(7) beige spleen cells and 2.5x10(6) purified wild-type splenic NK cells. Recipients infused with wild-type SPC exhibited significant augmentation of allograft survival compared with ATS controls. However, graft survival was reduced in recipients that were infused with spleen cells from beige mice compared with recipients infused with wild-type SPC (median survival time (MST): 38 vs. 92 days, P=0.02). In contrast, infusions of beige BMC augmented allograft survival as well as wild-type BMC (MST: 47 vs. 49 days, P=0.76). Furthermore, the ability of wild-type SPC to augment allograft survival was abrogated by the depletion of NK cells (MST=92 vs. 34 days, respectively, P=0.005). The co-infusion of beige SPC and purified splenic NK cells enhanced allograft survival as well as wild-type SPC (MST=56 days, P=0.65). Finally, recipients infused with purified NK cells did not experience increased graft survival compared to recipients that received no infusion (MST=29 vs. 33 days, respectively, P=0.6). Donor splenic NK cells are necessary, but not sufficient, for the extension of graft survival by infusion of donor splenocytes, suggesting that they may work in concert with another cell-type. In contrast, the extension of graft survival by donor bone marrow does not depend on the presence of donor NK cells.

Costimulation blockade, busulfan, and bone marrow promote titratable macrochimerism, induce transplantation tolerance, and correct genetic hemoglobinopathies with minimal myelosuppression.

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.