ALBERT

Description: Abstract 3283 Donor-derived regulatory T cells (Treg) and natural killer (NK) cells can respectively improve stem cell transplant (SCT) outcome by reducing graft versus host disease (GVHD) severity and exerting a graft-versus-leukemia effect. High frequencies of donor Treg are associated with less GVHD, and low doses of interleukin-2 (IL-2) can expand both NK and Treg after allogeneic SCT. To explore the feasibility of improving the quality of peripheral blood SCT donations, we evaluated the safety and the tolerability of ultra-low dose IL-2 administration to volunteers with the aim of preferentially expanding Treg and NK cells. Twelve healthy volunteers (mean age 34 years; range 22–57) received 0.1 or 0.2 million U/m2/day IL-2 subcutaneously for 5 days (NIH protocol 11-H-0268). Blood samples were collected before and 1, 2, 3, 4, 7 and 28 days after IL-2 injection. Samples were analyzed by multiplex techniques including whole transcriptome gene expression with HumanGene 1.0ST microarrays; serum levels of 69 cytokines and chemokines by Luminex assay; and lymphocyte phenotyping by flow cytometry, to comprehensively characterize the cellular and molecular immune response to IL-2 (“IL-2 immunome”). Treg subsets were determined within the CD4+ T cell population using FoxP3, Helios, CD45RA and CD31 to identify thymus-derived natural Treg (nTreg), induced Tregs (iTreg) and their recent thymic emigrants (RTE). NK cell subsets were determined within CD56+CD3- population using NKG2A, KIR2DL1, KIR2DL2/3, KIR3DL1 and CD57 to identify CD56bright, CD56dim NKG2A+KIR-, and CD56dim KIR+CD57+ cells. All subjects tolerated ultra-low dose IL-2 with minimal adverse events (mainly grade 1–2 injection site reactions). The fraction of FoxP3+Treg in CD4 rose significantly above baseline peaking at 4 days (3.7% vs 5.8%; p=0.0004) after the first dose of IL-2. Treg subset analysis demonstrated that the fraction of nTreg and RTE nTreg in CD4 expanded significantly in the lower dose cohort compared to the higher dose cohort (p=0.004 and p=0.005 respectively). %CD56bright NK significantly increased at 7 days (p=0.008), whereas CD56dimNKG2A+KIR-, and CD56dimKIR+CD57+ NK cells remained at baseline. The Ki67 proliferation marker further verified a significant in vivo expansion of CD56bright NK cells with ultra-low dose IL-2. Cytokine and chemokine profiling demonstrated significant increase circulating level of IP-10 (P=0.0018) through day 2 to 4 after IL-2 injections. In contrast, circulating levels of IL-2, IL-6, IL-10, IL-15 and IL-17 remained unchanged after IL-2 injection. Gene expression microarray studies revealed significant changes in 24 genes (P value 〈 0.1 corrected by false discovery rate (FDR) for multiple testing), including up-regulation of IL-2RA and FOXP3 as early as 2 days after IL-2 injections. Gene Set Analysis (GSA) revealed significant changes (P value 〈 0.1 after FDR) in innate immune response pathways, including Toll-like receptor signaling and interferon signaling. This is the first study to show that ultra-low dose IL-2 could be safely administrated to healthy volunteers to expand thymic-derived natural Treg and CD56bright NK cells. These results raise the possibility of using ultra-low dose IL-2 to boost Treg and NK cells in stem cell donors. Disclosures: No relevant conflicts of interest to declare.

Description: CD4+CD25+ regulatory T cells (Treg) have been hypothesized to control the development and progression of autoimmunity by suppressing autoreactive T cells. Treg are characterized by constitutive expression of membrane CD25 and intracellular expression of the transcription factor forkhead box (FOX) P3. FOXP3 and NF-AT1 have key roles in regulatory T-cell development and function: induction of FOXP3 in naïve T cells by gene transfer results in gain of a regulatory phenotype, and NF-AT1 modulates transcription of FOXP3. Treg display their suppressive properties on CD4+ CD25- T cells when activated via the T cell receptor or Toll-like receptor-2 (TLR2). Decreased numbers of FOXP3-positive regulatory Tregs have been associated with impaired immune homeostasis. Treg numbers are deficient in patients with active systemic lupus erythematosus, GVHD, and autoimmune hepatitis; in multiple sclerosis, decreased FOXP3 impairs Treg function. Acquired aplastic anemia (AA), the paradigm of immune mediated bone marrow failure syndromes, is characterized by immune-mediated destruction of hematopoietic stem cells. To examine expression of CD4+CD25+ T-cells in this disease, peripheral blood mononuclear cells from AA patients, sampled at diagnosis and prior to immunosuppressive therapy (n=18), were examined by flow cytometry: CD4+CD25hi+ T-cells were markedly reduced or absent in patients compared to healthy controls (n = 12, 0.08± 0.01 vs 0.4± 0.1%, p=0.001). CD4+CD25hi+FOXP3+ T-cells were also significantly lower in patients (0.05± 0.01 vs 0.32± 0.1, p=0.001). By immunoblot, we observed significantly decreased and often undetectable FOXP3 protein levels in CD4+CD25+ T cells from patients (n=8) compared to controls (n=6). Low FOXP3 protein levels correlated with decreased FOXP3 mRNA levels, as measured by RT-PCR and quantitative real-time PCR experiments (n=5, p=0.03). There were no differences in TLR2 in immunoblots between patients and healthy controls despite differences in FOXP3 expression. Patients (n=8) with low FOXP3 levels also showed decreased or absent NF-AT1 protein levels. These data collectively implicate a transcriptional mechanism for FOXP3 down-regulation. In confocal microscopy, purified CD4+CD25+ T cells from patients (n=5) showed undetectable NF-AT1 and FOXP3 levels as compared to controls (n=3). Four patients studied 3–6 months after first sampling and post-immunosuppressive treatment showed increased Tregs and FOXP3 expression; four further patients in complete remission after immunosuppressive treatment had increased CD4+CD25hi+ T-cells as compared to treatment-naïve patients. CD4+CD25+FOXP3+ regulatory T-cells are decreased in most patients with AA, probably through transcriptional regulation; decreased NFAT1 could explain low FOXP3 expression and Treg frequency. Treg employed as cellular therapy in a murine model of immune-mediated AA can prevent T cell-mediated marrow destruction (Chen J and Young NS, unpublished data). Treg defects are now implicated in autoimmune marrow failure in aplastic anemia.

Description: The primary granule proteins (PGP) neutrophil elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1 which can induce cytotoxic T lymphocyte (CTL) responses in chronic myeloid leukemia (CML). The relative contribution of PR3 and ELA2 to PR1 expression is not known. We previously found that higher levels of PR3 and ELA2 gene expression in CD34+ progenitor cells were associated with longer survival in CML patients. Eradication of leukemia depends on the elimination of leukemic stem cells which are thought to reside within the CD34+ progenitor cell pool. We therefore studied PGP expression and T cell response to PR1 in 23 CML patients and their HLA-identical family donors prior to T-cell depleted allogeneic stem cell transplantation (SCT) with T-cell add-back on day 45–100 post-SCT. CD8+ T cells and CD34+ progenitor cells were purified from mononuclear cells (MNC) of cryopreserved leukapheresis products from HLA-A*0201+ CML patients. Following reverse transcription of RNA from MNC and CD34+ cells, ELA2 and PR3 gene expression was measured using real-time quantitative polymerase chain reaction (RQ-PCR). To assess PR1-CTL responses, T2 cells were loaded with PR1 peptide (VLQELNVTV) at 0.1, 1, and 10μM for 2h, irradiated and subsequently co-cultured with CD8+ T cells for 3h. PR1-CTL response was measured as interferon-γ mRNA expression (relative to CD8) obtained by RQ-PCR. PR1-specific CTL responses were detected in 5/23 CML patients and 6/16 HLA-identical donors. In CML patients, pre-SCT expression of both PR3 and ELA2 in MNC was strongly correlated with the expression in CD34+ cells (p=0.009 and p=0.0006 respectively). There was an inverse relationship between PR1-CTL response in CML patients pre-SCT and PR3 or ELA2 expression (p=0.02 and p=0.01 respectively). This data suggest that both PR3 and ELA2 expression in CD34+ cells and their progeny are a potential source of PR1. However, high expression of these proteins may result in selective deletion of PR1 T cell clones. The presence of PR1-responses in HLA-identical donors was associated with an improved overall survival post-SCT. However, there was no impact of PR1-response in either CML patients pre-SCT or donors, on time to achieve molecular remission post-SCT but a greater proportion of patients whose donors did not have a PR1-response succumbed to fatal graft-versus-host disease. These findings support post-transplant vaccination strategies with PR1 peptide to eradicate minimal residual disease but in patients who are not eligible for SCT, the lower PR1 response in the presence of high PGP expression suggests that there is a risk that vaccines given to patients with minimal residual disease could induce tolerance. Figure Figure

Description: Donor lymphocyte infusions (DLI) following hematopoietic stem cell transplantation may reduce or control opportunistic infections and leukemia/lymphoma relapse, but the associated graft versus host disease (GvHD) limits the clinical success of this procedure. Since T cell immunotherapy may be a safer alternative to DLI we have now used a single T cell platform that mediates both antileukemic and antiviral activity. Autologous T cells modified to express CD19-specific chimeric antigen receptors (CD19.CAR) have had clinical activity against CD19-expressing malignancies, but it is unknown if similarly modified allogeneic T cells will be equally effective. Allogeneic virus specific T cells (VSTs) directed to cytomegalovirus (CMV), adenovirus (Adv), and Epstein Barr virus (EBV) have been shown to be safe and effective in preventing and treating life-threatening viral infections post HSCT. Therefore, we sought to determine whether allogeneic VSTs could be engineered to express CD19.CAR and would retain the safety and effectiveness of unmodified VSTs whilst gaining anti-tumor activity. VSTs were expanded ex vivo using antigen presenting cells engineered to express adenovirus and cytomegalovirus (using an Ad5f35 adenoviral vector expressing the CMV pp65 gene), and Epstein Barr virus (using EBV-infected lymphoblastoid cell lines) antigens. After 3 stimulations, the VST’s were modified to express CD19.CAR.28ζ using a retroviral vector encoding the CAR-CD19 receptor coupled to the CD28 co-stimulatory molecule and the T cell receptor zeta (ζ) chain. Nine CD19.CAR-modified virus specific T cell (CD19.CAR-VSTs) products were generated for infusion. All VST lines recognized at least one viral antigen as determined by Elispot or chromium release assays and 20% to 48% of cells expressed the CD19.CAR. All lines killed CD19-expressing cells in vitro. We treated nine patients with these CD19.CAR-VSTs, 3 months to 13 years after HSCT. Six patients received CD19.CAR-VSTs for relapsed disease and 3 patients received the T cells as adjuvant therapy to prevent viral infection and relapse after HSCT. Safety. There were no infusion-related toxicities. One patient presented with gastrointestinal symptoms following infusion subsequently determined to be unrelated to the T cells. Persistence. VSTs persisted a median of 8 weeks in the peripheral blood and up to 9 weeks at disease sites. In three patients (#1, #3 and #5), CD19.CAR signals were detectable in the bone marrow or the lymph nodes (44.8, 25.85, and 32 copies/1000 ng DNA) even when no signal was measurable in peripheral blood, indicating preferential accumulation of the infused T cells at the disease site. Anti-Tumor Activity. During the period of CD19.CAR-VST persistence, objective anti-tumor activity was evident in 2/6 patients with relapsed disease (patient # 1 had detectable blasts in the peripheral blood which disappeared within 1-2 weeks following infusion, patient # 2 had 16% circulating CLL cells which decreased within 2 weeks of T cell infusion) but disease recurred after 3 and 2 months, respectively. The two patients who received cells while in remission remain disease-free 〉3 and 〉9 months later. Anti-Viral Activity. In two patients with EBV reactivation, donor CD19.CAR-VSTs expanded concomitant with an increase in virus-specific T cell responses, and decreased viral load. A third patient had a rise in adenovirus specific VSTs during an episode of adenovirus associated diarrhea. Although the infection was controlled, there was no concomitant rise in CD19-CAR expressing T cells in this patient. No other patient had viral disease. In conclusion, allogeneic CD19.CAR-VSTs administered after allogeneic HSCT are safe and can exert both anti-tumor and anti-viral activity in the absence of GvHD. Earlier administration of CD19.CAR-VSTs after HSCT, when the host is lymphodepleted and the incidence of viral infection is higher, may allow these cells to better capture the potential advantages of native TCR stimulation (and associated co-stimulation) for expansion and persistence, and thereby produce a higher frequency of sustained tumor responses. Alternatively, intentional stimulation of the native TCRs by viral vaccines may produce equal benefit, with greater predictability. Disclosures: Savoldo: Celgene: Patents & Royalties, Research Funding. Heslop:Celgene: Patents & Royalties, Research Funding; Cell Medica: Patents & Royalties. Rooney:Cell Medica: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; Celgene: Patents & Royalties, Research Funding. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding.

Description: Abstract 3002 Adoptive transfer of CMV-specific T cells derived from adult CMV-seropositive (CMVpos) donors can effectively restore antiviral immunity after stem cell transplantation. However due to the absence of CMV antigen-specific memory T cells in cord blood (CB) and adult CMV-seronegative (CMVneg) donors, different culture systems are required to generate virus-specific T cells for adoptive transfer. With a novel protocol we have generated CMVpp65-specific T cells from CB and found that 15/15 CB T cell lines recognized atypical epitopes of pp65. We then explored the generation of CMV-specific CTL from CMVneg donors using a GMP-compliant methodology and studied the epitopes recognized. CD45RA+ naive T cells were selected from the peripheral blood of CMVneg donors and stimulated with pp65-Pepmix-pulsed dendritic cells with supplemented with IL-7, IL-12, and IL-15. For subsequent stimulations T cells were stimulated with pp65-Pepmix-pulsed EBV-LCL and IL-15 or IL-2. CMVpp65-specific T cells (CMV-CTL) expanded from 8 of 11 CMVneg donors were primarily CD8+ T cells (mean 71%). Naïve donor CMV-CTL secreted IFN- γ in response to pp65 peptides (mean 224; range: 38–611 SFC/1×105 cells) compared to irrelevant peptides (mean 12;Range 3–37) as measured in Elispot assays and lysed pp65-pulsed target cells (mean :48; range :15–70%) but not negative controls (mean 22; range 4–40%). These CMV-CTL derived from naive (but not memory) T cells recognized only novel and atypical pp65 epitopes (such as the HLA-A2-restricted epitopes LQTGIHVRV and MLNIPSINV) but not the typical HLA-A2-restricted epitope NLVPMVATV as confirmed by ELISPOT and multimer analysis. These results are similar to CB-derived CTL. Analysis of the avidity of naïve donor CTL specific for the atypical CMV epitopes revealed that the 1/2 maximum effective concentration was similar (mean: 600 pM) to CMVpos CTL recognizing typical epitopes (mean: 300 pM), and more avid than CMVpos CTL recognizing atypical epitopes (mean: 4 nM), highlighting the difference between naïve-derived and memory-derived CTL. TCR sequencing performed on T cells specific for typical (CMVpos) and atypical (CMVpos, CMVneg, and CB) epitopes revealed that CMVpos donor CMV-CTL recognizing typical epitopes were markedly more oligoclonal than CTL recognizing the atypical epitopes derived from CB, CMVpos, or CMVneg donors. To address the concern that atypical epitopes might not be naturally presented by CMV-infected cells and therefore not recognized by in vitro generated CTL, we tested whether CMV CTL (from CB, CMVpos, CMVneg) generated using CMV AD169-infected fibroblasts or CMV VR1814-infected DCs would recognize the same epitopes. As before, CMVpos CMV CTL recognized typical epitopes of pp65 while CB and CMVneg CMV CTL recognized only atypical epitopes, suggesting that the epitopes are naturally processed and presented by APCs, and that the atypical epitopes observed are not an artifact of using exogenous antigens like the pp65 Pepmix. Thus, despite their unusual repertoire, T cells derived from CB or CMVneg donors are likely to control CMV infection. These results reveal major differences in the naïve and memory CMV specific T cell repertoire that merits further exploration. Nevertheless, we demonstrated that atypical epitopes are naturally presented by CMV infected cells and we are now evaluating the clinical efficacy of these CTL in recipients of CBT. These studies should determine if naive T cells primed in vitro are able to persist and establish memory and virus protection in vivo. Disclosures: No relevant conflicts of interest to declare.

Description: Acute SR-GVHD occurs in approximately 15% of patients undergoing allogeneic hematopoietic stem cell transplant (HSCT), and is associated with a 70-90% long-term mortality rate. We previously reported that concomitant blockade of TNF-α and IL-2 pathways with infliximab combined with daclizumab have a synergistic therapeutic effect, with a high probability of complete resolution of SR-GVHD. Although various treatment modalities are effective in the treatment of SR-GVHD, minimal long-term follow up data exists for complete responders to second line treatments. Here we report long-term outcomes in a cohort of 23 subjects developing SR-GVHD treated with infliximab/daclizumab. A consecutive series of 141 patients with a variety of hematological and non-hematological malignancies as well as nonmalignant hematological disorders including severe aplastic anemia (SAA), paroxysmal nocturnal hemoglobinuria and pure red cells aplasia, underwent a reduced intensity allogeneic HSCT from an HLA identical or single antigen mismatched relative at a single institution between 2/2001 and 12/2008. Transplant conditioning consisted of cyclophosphamide (60 mg/kg days -7, -6) and fludarabine (25 mg/m2days -5 to -1) with or without equine ATG or 6-12 Gy of total body irradiation. GVHD prophylaxis was with cyclosporine with or without additional MMF or MTX. Twenty three patients (median age 35 years, range 13-65 years) developed SR-GVHD at a median of 28 days post transplant. SR-GVHD was defined as absence of response to at least 6 days of high dose methylprednisolone therapy. Following a diagnosis of SR-GVHD, patients received a combination of daclizumab (1mg/kg given on days 1, 4, 8, 15, 22), infliximab (10mg/kg given on days 1, 8, 15, 22), broad spectrum bacterial and anti-fungal prophylaxis, and had their methylprednisolone tapered to 1mg/kg/day. Combined cytokine blockade was highly active against SR-GVHD, with 21/23 (87.5%) patients achieving a complete response (CR), defined as total resolution of GVHD in all involved organ systems. All complete responders survived to hospital discharge. With a median follow-up of 9 years (range 5-10 years), 9/23 (39%) survive, including 6 patients without chronic GVHD whose immunosuppressive therapy (IST) has been discontinued and 3 patients with chronic GVHD (2 limited and 1 extensive) who continue to be tapered off IST. Fourteen of 21 patients with resolution of SR-GVHD died a median 173 days post transplant (range 67-1039 days), including 1 from complications related to recurrent SR-GVHD, 6 from progression of malignancy (all solid tumors), 2 from bleeding related to peptic ulcer disease and 5 from infectious complications including invasive fungal infection and CMV disease. A subgroup analysis showed 5/6 patients with SAA developing SR-GVHD had a complete response to combined infliximab/daclizimab. Remarkably, at a median 6 years follow up, 67% (4/6) of these SAA patients were long-term survivors. All these survivors have maintained normal blood counts and remained transfusion independent with 100% donor chimerism in myeloid and T-cell lineages. Conclusion Patients with SR-GVHD treated with infliximab combined with daclizumab had a high probability of achieving a complete response with nearly 40% of patients having long-term survival. This is the first report to show that long-term survival can be achieved in a substantial proportion of patients receiving combined IL-2 and TNF blockade for SR-GVHD. Disclosures: Off Label Use: Infliximab is FDA approved for the treatment of psoriasis, Crohn's disease, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis and ulcerative colitis. Daclizumab gained FDA approval for use in transplant rejection.

Description: Bone marrow stromal cells (BMSC, also known as bone marrow-derived “mesenchymal stem cells”) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage, BMSC for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following BMSC infusion. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were monitored for plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and after BMSC infusion. BMSCs were prepared from marrow aspirates from healthy volunteers with the expansion of 3 passages. Ten subjects were infused a fixed dose of 2 x 106 BMSCs /kg weekly for 3 doses. There was no treatment related toxicity (primary endpoint). Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first BMSC infusion (fig1). Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for tissue injury. The GVHD complete responders survived significantly longer (〉300 days vs a median of 33 days), had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts but there was no clear difference in natural or induced Tregs. Cytokine changes also segregated with survival. These results confirm that BMSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. However BMSC were ineffective in patients with more aggressive GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSC, requires a relatively intact immune system. Early detection and BMSC treatment appear important in patients with refractory GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic HSCT is a curative therapy for leukemia and other hematologic malignancies and disorders. Acute Graft-versus-Host Disease (aGvHD) is a significant cause of morbidity and mortality that limits the success of HSCT. No large analysis of this complication has been recently performed. Risk factors for aGvHD after HLA-matched sibling myeloablative unmanipulated HSCT were analyzed in 1960 adult (≥18 yrs) patients treated for AML (n=761), ALL (n=303), or CML (n=896) and reported to the CIBMTR registry from 1995–2002 by 226 centers worldwide. All patients received cyclosporine+methotrexate (CSA+MTX) alone (85%) or CSA+MTX+other agents (15%) for aGvHD prophylaxis. 635 (32%) patients developed grade II-IV aGvHD before day +100 post–HSCT. Outcome was measured as time from HSCT to onset of aGvHD Grade II-IV with death as a competing risk. Statistically significant risk factors for aGvHD in the univariate analysis were: Age ≥ 40 vs 〈 40 at HSCT, RR (95% CI, P) =1.35 (1.16–1.58, P=0.0001); Race, White/Black vs. Asian/Hispanic, RR=1.65 (1.31–2.08, P 0.05 to remove each clinical factor from the model. Significant independent predictors of aGvHD Grade II-IV risk were: Conditioning Regimen, CyTBI vs BuCy, RR (95% CI, P) RR=1.4, (1.2–1.7, P 80, RR=1.3 (1.05–1.5, P=0.014) and recipient/donor CMV status, at least one + vs −/ −, RR=0.8 (0.7–0.99, P=0.04). For pts ≥ 40 yrs at BMT, PB was not an independent risk factor for aGvHD Grade II-IV. There are modifiable risk factors for aGvHD Grade II-IV which include conditioning regimen, stem cell source (in younger patients) and CMV negative donors for CMV negative recipients. There are non-modifiable risk factors such as recipient age, race and gender and underlying diagnosis. Identifying patients at high risk for aGvHD Grade II-IV may allow for individualized risk modification and result in altering treatment strategies.

Description: NK cell alloreactivity in HLA-identical sibling stem cell transplantation (SCT) is not well understood. We previously showed that higher (greater than the median of 150/ ul) NK30 (absolute NK count on day 30 post transplant in recipient) correlated with a particular “favorable” KIR combination (2DL5A, 2DS1, 3DS1) in the donor. Both high NK30 and “favorable” KIR correlated with improved transplant outcome after T-depleted myeloablative sibling donor SCT for myeloid malignancies. We therefore studied the readout of NK cell subset ratio, CD107a degranulation level and different KIR receptor expression level within each NK subset among these patients. Cryopreserved mononuclear cell from 16 pairs of donor pre-transplant sample with his/her corresponding recipient day 30 post-transplant sample were thawed, recovered overnight and incubated with K562 cells at an E:T ratio of 5:1 togather with CD107a-FITC. The incubation product was stained with multicolor fluorochrome antibody cocktails. Events were acquired on an LSR-II flow cytometer. Data was analysed by Flowjo software and Student t-test was performed on those readouts previously mentioned within donor and recipient samples, each is further grouped by high versus low NK30, favorable vs unfavorable KIR and mild (grade 0, 1) versus severe (grade 2 and above) acute GVHD (aGVHD). In recipient day 30 post-transplant samples, the high NK30 group had significantly higher CD56+CD16+ NK subset ratio (p