ALBERT

Description: Donor lymphocyte infusions (DLI) can produce lasting remissions in patients with relapsed CML after allogeneic HCT, but are less effective in other non-CML diseases. We hypothesized that in vivo expansion of DLI may enhance their anti-leukemia effects. Mouse adoptive transfer experiments show that up-front lymphodepletion improves in vivo lymphocyte expansion by providing lymphoid space, eliminating host anti-donor immune reactivity and by decreasing competition for growth factors that promote lymphocyte expansion. In this clinical trial, lymphodepletion was achieved with IV cyclophosphamide (Cy) 50 mg/kg once on day −6 and fludarabine (Flu) 25 mg/m2 for five consecutive days (−6 to −2), a regimen we have shown previously to result in an in vivo surge of IL-15. DLI was given 48 hours after the last Flu dose and consisted of mononuclear cells (adjusted to a T-cell dose of 1 x 108/kg). Fifteen patients with relapsed non-CML disease received Cy/Flu/DLI as their first treatment of relapse between 2004 and 2006. CML patients who received the same cell dose (n=28) or non-CML patients (n=35) who received a higher dose consisting of 3 daily lymphapheresis products treated from 1993–2003 were used as controls. Since there was no difference in GVHD rates between CML and non-CML patients, they were grouped together as one control group. While most control patients did not have significant leukocytopenia and were treated as outpatients, the patients receiving Cy/Flu/DLI all became lymphopenic and neutropenic by the time DLI were infused and were treated in the hospital. Patients who received Cy/Flu/DLI developed significantly more overall (60% vs. 24%, P=0.01) and grade III-IV acute GVHD (47% vs. 14%, P= 0.01) compared to controls. The interval from DLI to grade III-IV GVHD was modestly shorter in Cy/Flu/DLI patients compared to controls (mean = 17 days vs. 34 days; P=.19). In Cy/Flu/DLI patients, blood lymphocytes were collected before and at 14 days, 28 days, and 2 months after DLI for immunophenotyping. Proliferating T-cells, as measured by expression of the Ki67 marker, were significantly increased 14 days after DLI (10.3±2.8%) compared to baseline pre-DLI (1.6±0.6%, P=0.012), and were already returning to baseline levels 28 days after DLI (3.5±1.2%, P=0.19). Despite aggressive therapy, 4 of the 7 patients who developed grade III-IV GVHD died from complications of GVHD, suggesting that Cy/Flu/DLI induces immune activation that is sufficiently potent to enhance toxicity. Therefore, a decreased DLI dose (half) is currently being used in subsequent patients. These data show “proof of concept” that DLI following lymphodepleting chemotherapy leads to in vivo lymphocyte expansion and results in more severe GVHD than when giving patients DLI alone. The ability of lymphodepletion to enhance the immune effects of DLI is promising if alloreactivity correlates with remission induction. This strategy warrants further study to understand if the increased severity of acute GVHD leads to more complete or prolonged disease control and whether the immune activation can be controlled with lower cell doses.

Description: Activation of Notch signaling regulates differentiation and homeostasis of hematopoetic stem cells. After stimulation, intracellular Notch is proteolytically released and by binding the CSL complex and co-activator MAML, and initiates transcription of downstream genes. We hypothesize that Notch is important for distinct stages of lymphoid development. Human cord blood CD34+ progenitor cells were transduced with retrovirus based eGFP-control, eGFP-Notch and Notch Dominant Negative/MAML (eGFP-DN) constructs. CD34+/eGFP+ were sorted and then co-cultured with the mouse embryonic liver cell line EL08.1D2 and exogenous human cytokines (IL-3. IL-7, IL-15, Flt3 ligand and c-kit ligand). As early as 48 hours after transduction, CD34+/Notch+ cells gave rise to population of lymphoid precursors CD34+CD7+CD10- (42±5% of all cells) while essentially no cells with this phenotype were detected with the control or DN construct. Proliferation of eGFP-Notch transduced cells in a 6-day thymidine incorporation assay was higher compared to eGFP-DN transduced cells (8410±839 vs. 1103±209 cpm; n=3; p=0.00005). Within 7 days 11±1.5% NK emerged from CD34+/Notch+cells compared to 0.8±0.2% of CD34+/eGFP+ control cells (n=5, p=0.0001). NK cell generation peaked at day 28 with a significantly higher expression of CD7 on NK cells (Notch: 75±5% vs. eGFP: 4.5±1%, n=5, p=0.00004), and no B lymphocytes were seen. Analysis of Notch induced NK cells demonstrated early expression of L-selectin and increased expression of CD45RA on all lymphoid progenitors. At 4 weeks, functional testing revealed reduced cytotoxicity against K562 (Notch: 37±0.5% vs. eGFP: 63.5±1.3%; n=7, p=0.007) suggesting immature function. CD34+/Notch+ derived NK lymphocytes also showed diminished acquisition of the lectin-type receptor NKG2A (Notch: 8.3±3% vs. eGFP: 27.4±4.5%; p=0.04) and killer immunoglobulin receptors (Notch: 2.2±0.5% vs. eGFP: 10.8±4: p=0.05). We next asked whether the Notch induced CD7+ precursor was NK restricted or a common NK/T cell precursor. After 5 weeks in culture, a distinct population of CD3+ T-cells emerged (Notch: 18±5% vs. eGFP: 1.6±0.2; n=5, p

Description: NK cell KIR interactions are among the variables known to affect clinical outcomes including relapse, graft versus host disease (GVHD) and survival after HCT. We hypothesized that T cells in graft sources available for HCT may affect KIR recovery and the therapeutic potential of KIR alloreactivity. We studied KIR reconstitution (the percentage of KIR+ NK cells measured by flow cytometry) in blood collected from recipients at day +100 after T cell deplete (TCD-BMT) and unmanipulated (U-BMT) unrelated BM transplants. We found that KIR reconstitution was suppressed compared to the healthy donors, significantly more so after U-BMT transplants (donor: 48.42 ± 2.35% KIR+ NK cells versus recipient: 26.74 ± 1.94, n = 36; P 〈 .001) than after TCD-BMT transplants (donor: 53.34 ± 3.25% versus recipient: 42.68 ± 3.32%, n = 38; P = .017), with P = .001 between the recipient groups. Additionally, multivariate Cox proportional hazards models showed that improved KIR recovery independently correlated with improved survival and that higher NK cell IFN-γ production independently correlated with more frequent acute GVHD in that patient cohort. These data suggested that T cell number in the graft affects KIR reconstitution and transplant outcome. We next examined other sources of hematopoietic cells in which T cell function may be suppressed either by growth factor mobilization (sibling donor unmanipulated peripheral blood: SibU-PB) or the innate naivety of the T cells (umbilical cord blood: UCB). KIR+ NK reconstitution on recovering cells at day +100 after all HCT graft types was significantly less than that on normal donor cells (normals 55.33 ± 1.73%, n = 124; all P 〈 .0006). U-BMT recipients had significantly lower KIR+ NK recovery (27.31 ± 2.06%, n = 36 vs. SibU-PB: 37.58 ± 3.29%, n = 29; TCD-BMT: 42.68 ± 3.32%, n = 38; or UCB, 37.99 ± 2.54%, n = 49) when compared to all other transplant types. The highest absolute T cell inoculum, found in SibU-PB, showed KIR reconstitution similar to that of TCD-BMT, which had the lowest T cell content (p=0.29), perhaps due to the lower alloreactivity of the Sib grafts and to the G-CSF-priming which preferentially mobilizes T cells with a suppressive phenotype. Similarly, KIR reconstitution was better after UCB compared to U-BMT (P = .0027), possibly due to the more permissive interactions with naive T cells. These results suggest that reduced T cell number after T cell depletion, suppressed T cells found after growth factor mobilization, or naive T cells present in UCB grafts enhance in vivo KIR reconstitution after allogeneic HCT when compared to unmanipulated marrow grafts. Such enhanced KIR reconstitution may have clinical consequences. Graft T cells may directly compete for cytokines and growth factors, or may be a surrogate marker for other transplant factors such as the development of GVHD and the requirement for intensive post-transplant immunosuppression. Understanding these interactions will allow judicious selection of hematopoietic cell source to select for enhanced KIR recovery. For example, among unrelated unmanipulated donor grafts, KIR+ NK recovery was significantly better using UCB than adult donors and further investigation may show that this is advantageous to improve clinical outcomes.

Description: Although unrelated hematopoietic cell transplantation (HCT) is curative for many hematologic malignancies, complications and relapse remain challenging obstacles. Natural killer (NK) cells, which recover quickly after transplantation, produce cytokines and express killer immunoglobulin-like receptors (KIRs) that regulate their cytotoxicity. Some clinical trials based on a KIR ligand mismatch strategy are associated with less relapse and increased survival, but results are mixed. We hypothesized that T cells in the graft may affect NK cell function and KIR expression after unrelated transplantation and that these differences correlate with clinical outcomes. NK cell function was evaluated using 77 paired samples from the National Marrow Donor Program Research Repository. Recipient NK cells at 100 days after both unmanipulated bone marrow (UBM) and T-cell depleted (TCD) transplants were compared with NK cells from their healthy donors. NK cells expressed fewer KIRs and produced more interferon γ (IFN-γ) after UBM compared to TCD transplants. Multivariate models showed that increased NK cell IFN-γ production correlated with more acute graft-versus-host disease (GVHD), and decreased KIR expression correlated with inferior survival. These results support the notion that T cells in the graft affect NK cell reconstitution in vivo. Understanding these mechanisms may result in strategies to improve clinical outcomes from unrelated HCT.

Description: Donor lymphocyte infusions (DLIs) can produce lasting remissions in patients with relapsed chronic myeloid leukemia (CML), but are less effective in non-CML diseases. We hypothesized that lymphodepletion, achieved with cyclophosphamide (Cy) and fludarabine (Flu), would promote in vivo expansion of the infused lymphocytes enhancing their immunologic effects. Fifteen patients with relapsed non-CML disease who received Cy/Flu/DLI were compared with 63 controls who received DLI without chemotherapy. Only the patients receiving Cy/Flu/DLI became lymphopenic at the time of DLI. Compared with controls, patients who received Cy/Flu/DLI developed significantly more grades II to IV (60% vs 24%, P = .01) and grades III to IV acute graft-versus-host disease (GVHD) (47% vs 14%, P = .01) with greater GVHD lethality. In Cy/Flu/DLI patients, T-cell proliferation was elevated at 14 days after DLI. Although these data suggest that chemotherapy-induced lymphodepletion enhances activation of donor lymphocytes, the toxicity needs to be managed before testing whether better disease control can be achieved. This trial was registered at www.clinicaltrials.gov as no. NCT00303693 and www.cancer.gov/clinicaltrials as no. NCT00167180.