Alloreactive Cells Research Articles

Translational Research Efforts in Biomarkers and Biology of Early Transplant-Related Complications

In the time since the first allogeneic hematopoietic stem cell transplantation (HSCT) was performed over 40 years ago, this life-saving procedure has been used increasingly for patients with hematologic, metabolic, and malignant diseases. Despite major advances in our understanding of the immunologic processes (both beneficial and injurious) that are associated with HSCT and improvements in supportive and critical care medicine, successful outcomes are still limited by several serious complications. As such, the establishment of effective therapeutic strategies for these complications will be crucial as increasing numbers of high-risk transplants are performed each year. The development of such approaches is fundamentally dependent upon a basic understanding of pathophysiologic mechanisms of disease and also on our ability to successfully translate these insights back to the bedside. This brief review will highlight breakthroughs in translational research endeavors that have paved the way for the development of novel strategies intended to change the standard of care and optimize outcomes for patients in whom allogeneic HSCT offers the only hope for a cure.

Determination of donor-derived killer immunoglobulin-like receptor (KIR) by sequential genotyping in hematopoietic stem cell-transplanted patients

Donor killer immunoglobulin-like receptor (KIR) and KIR–ligand mismatch is considered vital in clarifying the mechanism of natural killer (NK) cell alloreactivity in hematopoietic stem cell transplantation (HSCT). In practical terms, however, it may be difficult to analyze the KIR genotype of donor cells directly as all donor cells are used for the transplant rather than for research purposes. To accurately estimate donor KIR genotype, we determined recipient KIR genotyping sequentially, at a minimum of two time points, using 19 KIR-specific primers in 10 patients who underwent HSCT. Among 10 patients, four had a KIR–ligand mismatch in the graft versus host direction. Sequential KIR genotyping showed the genotype changes at the time of engraftment (donor-derived) as well as relapse (recipient-derived). Our results highlight the utility of sequential KIR genotyping to better understand ligand–ligand, KIR–KIR, or ligand–KIR mismatches. Further studies, including a functional assay of NK cells may clarify the underlying mechanism of KIR ligand–donor KIR mismatch in HSCT.

Everolimus and Mycophenolate as GvHD Prophylaxis Results In Improved Control of Advanced Disease and Reduced Bacterial and Fungal Infectious Complications After Allogeneic Stem Cell Transplantation

AbstractAbstract 1264Tolerance induction together with sustained disease control remain to be critical for longterm survivors after allogeneic hematopoietic cell transplantation (HCT). Regulatory T cells (Tregs) are involved in the regulation of graft versus host disease (GvHD) and graft versus leukemia effect (GvL). Although calcineurin inhibitors are frequently being used for the prevention of GvHD, tolerance induction might be hampered in HCT patients due to deteriorated Treg function. Data from animal models of HCT suggest that inhibition of the mammalian target of rapamycin (mTOR) results not only in suppressed T cell alloreactivity but also in sustained Treg function. On the other hand severe reduction of T cell alloreactivity might result in diminished control of especially advanced malignant disease. To explore the clinical efficacy of a calcineurin-free GvHD prophylaxis regimen, we initiated a phase I/II monocenter trial using everolimus and mycophenolate-sodium (MMF-Na) as GvHD prophylaxis in patients undergoing allogeneic HCT with peripheral stem cell (PBSC) grafts after reduced toxicity and standard conditioning. 28 patients were included (median age: 49,2 years, range: 21–65). The diagnoses were: AML/MDS (n=16), ALL (n=3), CML/MPS (n=3), T-PLL (n=1), NHL/CLL (n=6). At the time of transplantation 22/28 (78%) patients were at high risk (not in CR1/CP, untreated) for early relapse. Conditioning included fludarabin based reduced intensity (n=24) or standard regimens containing busulfan (n=2) or clofarabine (n=2). Four CR1 patients received additional alemtuzumab (total 10mg) for GvHD prophylaxis. PBSC grafts were obtained from unrelated (n=20) and related (n=8) HLA-matched donors. No graft failure occurred. Engraftment kinetics for myeloid cells were normal, patients without alemtuzumab showed rapid reconstitution of the T cell compartment with median cell counts of >200 CD4+ cells/μl at day +30 together with complete donor chimerism in 15/18 evaluable patients. No grade IV/V toxicities (according to CTC criteria) were observed due to the study medication. After a median follow up of 9 months, 3 relapses of 24 patients with CR after HCT (12,5%) occurred, of those one AML patient could be salvaged with withdrawal of immunosuppression. Nine patients have died. The causes were underlying malignant disease (n=2), GvHD (n=2), viral infections (n=3 with two cases of HHV6 associated encephalitis), post-surgery thrombembolism (n=1), and one unknown. Treatment related mortality after 100 days is 14,2%, after 1 year 21,4%. Due to the early recovery of T cell immunity mild forms of acute skin GvHD were common early after reconsitution, while acute GvHD Grade III-IV could be observed in 8/26 patients. Chronic GvHD occurred in 15/22 patients (68%) with moderate and severe forms in n=10 and n=3 patients, respectively. Cytomegalovirus (CMV) reactivation could be seen in 5/20 patients at risk, while no CMV disease developed. Importantly, in the first year after HCT no severe bacterial or fungal infections were observed even in cases with prolonged everolimus treatment. The whole cohort experienced a median overall survival of 20 months, median progression free survival was 19 months. In conclusion, GvHD prophylaxis with everolimus and MMF-Na is feasible but results in an increased frequency of moderate chronic GvHD without major bacterial and fungal infectious complications. Since this sustained alloreactivity might reduce the risk of relapse this regimen could be suited for patients undergoing HCT with advanced or uncontrolled malignant disease. Disclosures:No relevant conflicts of interest to declare.

Full Haplotype Mismatch Stem Cell Transplantation: Myeloablative with T-Cell Depletion Vs. Reduced Intensity Conditioning without T-Cell Depletion

AbstractAbstract 3533Some groups have continuously tried to reveal the possible role of full-haplotype mismatch stem cell transplantation (SCT) in patients who lack a compatible donor. However, the long-term event-free survival (EFS) rate after high-grade mismatch SCT using conventional myeloablative conditioning with T-cell depletion (TCD), even in complete remission (CR), is still only in the range of 10–30%. Most patients who were transplanted had heavily pretreated acute leukemia. The most important obstacle to overcome is the high rate of infection-related mortality associated with delayed immunological recovery in the setting of full-haplotype mismatch SCT, specifically by way of complete depletion of donor T cells in the graft. In contrast, by using unmanipulated donor cells and less aggressive conditioning regimens without TCD, most groups in Asia have reported EFS >40%. We investigated the role of reduced-intensity conditioning without TCD in HLA-mismatched related-donor SCT, specifically in patients with high-risk acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) who received unmanipulated granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). SCT was performed in 32 consecutive patients. We compared two different protocols: one with more intensive conventional conditioning with TCD (group 1), and one with reduced intensity without TCD (group 2). The protocols differed between the two groups with respect to total body irradiation (1200 cGy in group 1 vs. 800 cGy in group 2) as well as busulfex, fludarabine, and anti-thymocyte globulin (ATG; thymoglobulin, Genzyme) treatments. No patient in group 1 received post-transplant graft versus host disease (GvHD) prophlyaxis or G-CSF. In contrast, all patients in group 2 received 1.25 mg/kg/day ATG for 4 consecutive days, together with our standard GvHD prophylaxis regimen of methotrexate (5 mg/m2 intravenous bolus on days 1, 3, 6, and 11) and tacrolimus starting at day –1. All patients received G-CSF-mobilized PBSCs. The median number of CD34+ cells infused was 14.2 (range, 4.3–20.6) × 106/kg for group 1 and 6.0 (range, 3.6–8.5) × 106/kg for group 2. The median numbers of CD3+ cells infused were 0.03 (range, 0.015–0.057) × 106/kg and 870 (range, 84–1260) × 106/kg, respectively. G-CSF was administered subcutaneously to all patients in group 2 at a dose of 5 mg/kg/day, from day 7 after transplantation until neutrophil recovery. GvHD, relapse, non-relapse mortality (NRM), overall survival, EFS, reconstitution of immunity, and natural killer (NK) cell alloreactivity were compared between the groups. The median patient age was 33 (range, 17–48) years for group 1 and 39 (range, 19–63) years for group 2. The median follow-up for surviving patients was 18 months (range, 8–100). The majority of patients had intermediate or unfavorable cytogenetic features. The transplanted patients were all successfully engrafted. The median times to neutrophil (>0.5 × 109/kg) and platelet (>20 × 109/kg) recovery were 12 and 13 days in group 1, and 11 and 10 days in group 2. The overall rates of acute and chronic GvHD were 50% and 33%, and 80% and 92% in groups 1 and 2, respectively. Of note, NRM differed significantly between the groups: 46.2% in group 1 vs. 9.5% in group 2 (P = 0.0014). The estimated probability of EFS at 2 years was 15.3% for group 1 vs. 59.2% for group 2 (P = 0.05). We did not find NK alloreactivity in any of the patients, based on the NK-killer cell immunoglobulin-like receptor (KIR) disparity between donor and recipient. However, we noted a significant difference in EFS between C1/C1 homozygotes and C1/C2 heterozygotes according to the donor NK-KIR ligands (P = 0.0175). Recovery of CD4+ cell numbers at 3 months after SCT showed a markedly different pattern between the groups, with a median of 4 cells/μl (range, 2–7) in group 1 vs. 311 cells/μl (range, 56–1226) in group 2. Our findings suggest that full-haplotype mismatch SCT using a Korean-adapted protocol is a feasible therapeutic strategy for patients with high-risk AML/MDS, specifically in CR, provided that there is a further defined plan for the investigation of NK/T-cell alloreactivity. Disclosures:No relevant conflicts of interest to declare.

Killer Cell Immunoglobulin-Like Receptor (KIR) Ligands Predict Outcomes of Autologous Stem Cell Transplantation for AML

AbstractAbstract 3571The idea of natural killer (NK) cell alloreactivity after allogeneic stem cell transplantation (SCT) was developed from reports of patients with acute myelogenous leukemia (AML). However, little definite information exists about the killer cell immunoglobulin-like receptor (KIR) and its ligand activity in autologous SCT. Since whether autologous NK cells are involved in the eradication of leukemia cells is still unclear, we focused on whether autologous NK or CD8+ T cells are stimulated after myeloablative conditioning, according to the ligand match. In light of a previous report that KIR–HLA combinations have effects in lymphoma and solid tumors, we undertook this retrospective study of KIR–HLA interactions. To examine the impact of autologous KIR–HLA ligands, we analyzed the HLA epitopes in patients with adult AML who benefited from autologous SCT (n = 164). The median age of the patients was 34 years (range, 15–64) and the subjects consisted of 63 females and 101 males. All patients received our modified TAM conditioning, which consisted of fractionated total body irradiation (12 Gy, five fractions in 3 days) from day –8 to –6, followed by intermediate-dose cytarabine (Ara-C; 1.5 g/m2 over 3 h every 12 h for six doses) from day –5 to –3, and melphalan (100 mg/m2 over 30 min) on day –2 only. Following consolidation chemotherapy, we collected peripheral blood stem cells after administering granulocyte colony-stimulating factor in all patients, except for 17 poorly mobilizing patients who received peripheral blood stem cells plus bone marrow together. The median infused CD34+ cell dose was 4.2 × 106/kg (range, 0.4–40.7). Finally, the effects of the KIR genotype compared with HLA ligands on the autologous SCT outcomes were examined in 81 patients who were typed for the presence or absence of 19 different KIR genes and pseudogenes using a polymerase chain reaction-based sequence-specific primer method with the Pel-Freez kit, according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). KIR and HLA typing are defined by two subgroups of KIR ligands called C1 and C2. Furthermore, we divided C1/C1 homozygotes into two different KIR ligand patterns according to the HLA typing results: C1 homozygote homodimers (such as HLA-Cw3 and -Cw3; n = 32) and C1 homozygote heterodimers (such as HLA-Cw3 and -Cw8; n = 71). Fifty-three patients were typed as C1/C2 heterozygotes. The median follow-up for surviving patients was 65 months (range, 5–121). The majority of patients had intermediate (n = 90) or unfavorable (n = 41) cytogenetic features. At present, 66 patients (40%) have relapsed, and 94 patients (57%) have survived, while 10 (6%) died due to non-relapse causes. The Kaplan–Meier estimated 5-year overall and event-free survival (EFS) rates were 55% (95% CI, 50–60%) and 52% (95% CI, 48–56%), respectively. According to the consistent results of analyses of the HLA epitopes and genotypes, HLA ligand C1/C2 heterozygotes were compared with C1/C1 homozygotes. Heterozygotes, defined as KIR-HLA ligand matches, tended to have a better long-term EFS (P = .0636) and had a significantly lower relapse rate (P = .031). The number of infused CD34+ cells at the time of autologous SCT (P = .0279), FAB subtypes (M2 vs. other subtypes) (P = .0375), and risk grouping by cytogenetics (P = .0005) were other factors showing statistical significance on EFS and relapse. Since our findings are based on a single disease entity (i.e., adult AML), they suggest that autologous KIR–HLA ligands are, at least in some ways, critical for the influence of autologous NK immunomodulation on antileukemic effects, especially considering the impact of activating KIR–ligand activity on the long-term survival. Therefore, the identification of “ receptor–ligand ” effects could be useful as a prognostic marker and may be important in predicting patients who can expect long-term leukemia-free survival after autologous SCT for AML. Disclosures:No relevant conflicts of interest to declare.

Total Body Irradiation Creates a Proinflammatory Milieu That Is Required for Minor, but Not Major, Mismatch Graft-Versus-Host-Disease.

AbstractAbstract 3736 BackgroundGraft-versus-host disease (GVHD) is a potentially fatal complication following allogeneic bone marrow transplantation (BMT). GVHD is characterized by three phases: (1) recipient tissue injury mediated by the conditioning regimen of irradiation and/or chemotherapy; (2) donor cell priming and activation; and (3) effector destruction of target tissue such as the intestine, liver, skin, and lung. There is some evidence to suggest that the second and third phases are dependent on the first, however, this has not been rigorously tested. Therefore, we developed major and minor mismatch models of GVHD and used immunodeficient recipients that readily accept allografts to analyze the requirement of irradiation conditioning on GVHD initiation and development.Experimental Design Wild-type (WT) B6 (H-2b) recipients received major mismatch (Balb/c, H-2d), minor mismatch (129, H-2b), or syngeneic (B6) BMTs. Recipients were lethally irradiated, and 24 hours later received an intravenous infusion of 5 × 106 bone marrow cells and 30 × 106 splenocytes, as a source of mature T cells. In adoptive transfer (AT) experiments, B6 Rag1−/− or B6 Rag2−/−/Pfp−/− (perforin deficient) recipients received 30 × 106 splenocytes. Data are represented as mean ± SEM. ResultsIn lethally irradiated WT recipients, Balb/c→B6 caused severe GVHD that was fatal in all recipients by 24 days after BMT. 129→B6 led to a milder GVHD, where 50% of recipients survived to 35 days after BMT, and a few survived long-term (>100 days). Both major and minor mismatch GVHD were characterized by 15–25% weight loss, clinical symptoms including decreased activity, hunched posture, ruffled fur, and hair loss, and target organ histopathology. To test the requirement of irradiation conditioning in GVHD, we transferred Balb/c splenocytes into unconditioned B6 Rag1−/− recipients. No signs of GVHD developed. However, donor T cells were virtually undetectable 5 weeks after AT, indicating graft rejection. To test if this was due to recipient natural killer (NK) cells, a major mismatch AT was performed into B6 Rag2−/−/Pfp−/− recipients, which lack fully functional NKs. Balb/c→B6 Rag2−/−/Pfp−/− resulted in GVHD, including 10–15% weight loss, clinical symptoms, and target organ histopathology, although the disease was not as severe as that following lethal irradiation of WT recipients, consistent with a facilitative role for conditioning in disease progression. In contrast to the above, a 129 splenocyte AT did not cause GVHD in Rag1−/− or Rag2−/−/Pfp−/− recipients, despite donor T cell engraftment. This suggested that conditioning was required for minor mismatch GVHD. To test this, we sublethally irradiated Rag1−/− recipients prior to 129 AT. This resulted in GVHD, with 10–15% weight loss, clinical symptoms, and histopathology. At day 7 after AT, serum IFNγ, TNF, and IL-6 were significantly greater in mice that received irradiation and AT (compared to AT alone, p<0.05). Donor cells within spleens of mice that received irradiation and AT had a reduced fraction of Foxp3+/CD4+ (9.5%±2.3) compared to AT alone (16.8%±1.6, p=0.06), and increased intracellular CD8+ IFNγ expression (%IFNγ+:47.7±2.4 vs. 41.6±1.8, p<0.05; MFI:15722±2003 vs. MFI:8025±319, p<0.05). To determine whether more alloreactive donor cells were primed after irradiation, an in vivo killing assay was performed with recipient-specific targets. Combined irradiation and AT resulted in 66.3±13.5% killing efficiency while AT only had an 18.9±11.7% killing efficiency. ConclusionsWe conclude that irradiation conditioning is required for minor, but not major mismatch GVHD. The conditioning regimen creates the necessary proinflammatory milieu to prime sufficient numbers of alloreactive cells for GVHD. While this milieu can enhance development of major mismatch GVHD, it is not required for development of disease. Disclosures:No relevant conflicts of interest to declare.

CASPALLO: Phase I Clinical Trial of Allodepleted T Cells Transduced with Inducible Caspase 9 Suicide Gene After Haploidentical Stem Cell Transplantation

AbstractAbstract 559Adverse effects from cellular therapies may be prolonged and progressively worsen as cells expand and persist long-term, unlike most small molecule toxicities, which diminish following drug withdrawal. “Suicide genes” can be introduced into administered cells and activated – generally by a small molecule pro-drug - in the event of adverse effects. Although one such approach using a transferred Herpes Simplex Virus thymidine kinase (HSV-tk) gene may provide effective control, its mechanism of action requires interference with DNA synthesis. As a consequence, cell killing may be protracted over several days, with an even longer delay in clinical benefit. Moreover, an anti-viral therapeutic pro-drug (such as ganciclovir) is required for cell elimination, removing this class of agents from the therapeutic repertoire. Finally, HSV-tk is virus-derived and hence potentially immunogenic. We now report the clinical evaluation of a human suicide gene, inducible Caspase-9 (iCasp9), which is designed to interface with the physiological apoptotic pathway. To generate the iCasp9 molecule we modified human caspase 9 to dimerize and activate upon exposure to a synthetic, otherwise bioinert, small molecule dimerizing agent, AP1903.We infused iCasp9-expressing T lymphocytes, in an effort to enhance immune recovery and reduce infection/relapse following transplantation of HLA-haploidentical hemopoietic (CD34+) stem cells as treatment for high-risk, relapsed leukemia. The infused T cells were first depleted of alloreactive progenitor cells after stimulation with recipient irradiated, EBV-transformed lymphoblastoid cells (40:1) for 72 hrs, and subsequent exposure to a CD25-directed immunotoxin (RFT5-dgA). The allodepleted cells were then transduced with a retroviral vector encoding the iCasp9 suicide gene and a selection marker (DCD19), which allowed enrichment to >95% purity.Four subjects received 1–3 x106 gene-modified T cells/kg. Forced expression of a transgenic caspase-derived molecule did not preclude in vivo survival or expansion of infused T cells, which became detectable by flow cytometry (CD3+DCD19+cells) and by Q-PCR (for iCasp9) within 7 days of infusion. By day 14 these cells expanded to a median cell number/μ l of 175 (range 2–348) (dose level 1) and 49 (range 4–93) (dose level 2). The iCasp9+ T cells contained both CD4+ and CD8+ subsets, were viral-reactive (CMV, EBV, and ADV) and polyclonal, and have now persisted beyond 240 days. Three of the 4 subjects subsequently developed grade I/II acute GvHD of skin, one of whom also had a rising bilirubin, attributed to liver GvHD. As per protocol, these subjects received a single dose of dimerizer agent. Within 30 minutes of completing AP1903 administration, we observed a circa 90% reduction of transgenic T cells, as assessed by flow cytometry for CD3+CD19+ T cells and by Q-PCR amplification for iCasp9. This effect was followed within 36 hrs by resolution of all aGvHD, showing that the iCasp9 transgene can be functional in vivo and can rapidly deplete sufficient T cells to control GvHD. Of note, the residual allodepleted T cells were no longer associated with GvHD but were still able to re-expand within 21 days (median CD3+CD19+ cells/μ l: 77 (range 38–87)), and contained subpopulations that preserved reactivity to viruses (CMV) and fungi (Aspergillus fumigatus), as assessed by IFN-γ production.In conclusion, administration of small numbers of iCasp9+ allodepleted T cells may produce CD4+ and CD8+ T-cell reconstitution after haplo-identical, CD34+ SCT, while administration of a small molecule dimerizer agent has rapidly ablated residual allo-reactive T cells and abrogated early GvHD, whilst preserving anti-viral specificity.Supported by NIH-NHLBI U54HL081007 Disclosures:No relevant conflicts of interest to declare.

Post-Transplant Cyclophosphamide Treatment Ameliorates Experimental Gvhd While Permitting Lymphopenic Expansion of Non-Host Reactive Donor T Cells.

AbstractAbstract 3751Following myeloablative as well as reduced intensity conditioning regimens, recipients of allogeneic T cell replete (TCR) HLA matched sibling)or unrelated hematopoietic stem cell transplants (HSCT) experience significant acute as well as chronic graft vs. host disease (GVHD). Recent studies have reported that post-transplant cyclophosphamide (PTC) can function as an effective single agent for prophylaxis of acute and chronic GVHD (Biol. Blood Marrow Transpl. 16:482, 2010; Blood. 116:3224, 2010). To investigate the fate of non-host alloreactive donor cells, which are critical to providing immune reconstitution post-transplant, T cell replete HSCT were performed between MHC-matched allogeneic donor–recipient pairs. C3H.SW (H2b) mice were lethally conditioned 4 hrs prior to receipt of B6-CD45.1 (H2b) T cells and bone marrow. Recipients developed severe GVHD as assessed by weight loss (Fig. 1A) and clinical analyses during the first 4–5 wks post-transplant. Recipients had inverted CD4/CD8 ratios as well as an activated phenotype (CD44hiCD62Llo), severe B and T cell lymphopenia, and virtually undetectable thymic tissue. A single dose of post-transplant cyclophosphamide at day 3 (66mg/kg/body weight) blocked onset of weight loss and clinical signs of GVHD as well as lethality in C3H.SW recipients, consistent with the reduction of alloreactive anti-recipient T cells. In contrast to non-cyclophosphamide treated animals, treated recipients expressed typical CD4/CD8 ratios, significant numbers of spleen and lymph node cells (Fig. 1B) and readily detectable thymi 7–8 weeks post-HSCT. Notably, the majority of T as well as B cells in the periphery of these recipients exhibited a non-activated phenotype and were derived from donor bone marrow. Together with the observation that approximately 50% of T cells exhibited a CD44hiCD62Llo phenotype characteristic of TN, the findings are consistent with de novo derived lymphopoiesis in the thymus and marrow of post-transplant cyclophosphamide treated recipients. To assess the impact of D.3 cyclophosphamide treatment on a non-host reactive T cell population, B6 OT-I CD8 T cells (Vα2/Vβ5 TcR) were added (1-2×106) to the donor inoculum. In non-PTC treated recipients, low numbers of OT-I T cells (below input levels) were detected by 3–4 weeks post-HSCT. In contrast, in PTC-treated recipients, significant numbers of OT-I CD8 T cells were present in the recipient spleen and lymph nodes (Fig. 1C), consistent with the finding of CFSE labeled OT-I cells exhibiting division following cyclophosphamide treatment. Notably, the overall numbers of OT-I CD8 T cells in cyclophosphamide treated recipients was greater than the input levels, indicating that these cells a) survived post-cyclophosphamide treatment and b) underwent lymphopenic expansion. Consistent with these findings, CFSE studies at 72 hrs post-HSCT illustrated OT-I T cells exhibited low proliferation vs. a population of presumed B6 anti-C3H.SW alloreactive T cells. We therefore conclude that non-host reactive donor T cells undergo sufficient repair following cyclophosphamide induced alkylation to enable greater survival in contrast to rapidly dividing anti-host (GVHD) reactive populations. Vaccination studies in cyclophosphamide treated and non-treated HSCT recipients are being performed using heat shock fusion protein-transfected tumor cells to investigate the capacity to elicit anti-tumor (i.e., GVL) responses in the presence and absence of GVHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

SiglecH Positive Myeloid Cells Mobilized by Pegylated Murine GM-CSF Have Potent Allo-Suppressive Properties In Vitro.

AbstractAbstract 1459Granulocyte-macrophage colony stimulating factor (GM-CSF) is a growth factor regulating proliferation and differentiation of hematopoietic stem and progenitor cells. Pegylation of murine GM-CSF (peg-mGM) has been shown to prolong its half-life. We have observed that patients transplanted with stem cell products mobilized by GM-CSF have a lower incidence of acute GvHD (Devine et al. BMT 2005), we hypothesized that peg-mGM mobilized stem cell products may be protective from acute GvHD in MHC mismatched murine models.C57/Bl6 (B6) mice treated with peg-mGM (5mcg IP qd × 4) resulted in increased CFU-C by 26.5 fold compared to PBS treated mice (n=6, 95%CI 10.25–42.73). In addition, peg-mGM increased CD4+FoxP3+ Tregs in both blood and spleen by 2 fold compared to PBS (p<0.05). Importantly, the function of these peg-mGM Tregs, as measured in a mixed lymphocyte reaction (MLR) was found to be equivalent to Tregs from PBS treated control mice (t-test, n=3, p=0.27). Furthermore, we have observed an increased expression of Ki67 in murine Tregs in vivo in response to peg-mGM treatment consistent with peripheral expansion of Tregs. We hypothesized that a peg-mGM mobilized product would cause less graft versus host disease (GvHD). We have assessed the in vivo impact of peg-mGM on GvHD, by using the B6 to B6D2F1 acute GvHD model. B6 donors were treated with peg-mGM or PBS for 4 days; on the 5th day splenocytes were isolated. An equivalent dose of CD3+ T-cells along with congenic T cell depleted bone marrow was administered to recipients (n=5/group) that had undergone myeloablative conditioning (11 Gy TBI). We observed less weight loss and improved survival in the peg-mGM treated group (Log-rank chi-square 53.22, df=9, p<0.001). This effect may be explained by an increase in Tregs delivered in the peg-mGM group but also potentially to additional accessory cells that could be present in the graft and mediating an allo-suppressive response. We have phenotyped cells mobilized by peg-mGM and have observed a novel cellular phenotype consistent with a monocyte/macrophage mobilized in the peripheral blood that is SiglecH+, B220-, Ly6c+, CD11b+, GR-1 intermediate, and F4/80+/−. This subset appears within 2 days of the start of peg-mGM treatment and was not observed in mice mobilized with PBS, G-CSF or plerixafor. To evaluate the function of this novel population and its potential suppressor properties we performed an MLR. B6 mice were mobilized with peg-mGM for 4 days, on the 5th day whole splenocytes were isolated and single cell sorted for SiglecH+B220- population or B220+SiglecH- (control cells). These cells were incubated with irradiated Balb/C splenocytes (stimulators) and CFSE labeled congenic B6 CD4+CD25- T-conventional (Tcon) cells (responders) at varying ratios of stimulators:responders:suppresors. Freshly isolated naive Tregs were used as the positive control. At the end of 6 days of incubation wells containing the SiglecH+B220- cells at 1:1:1 or 1:1:2 ratios suppressed allo-reactive Tcon cells as potently as Tregs at a 1:1:1 ratio (t-test, n=3, p=0.49 and p=0.98 respectively) compared to no suppressor controls (t-test, n=3, p=0.02 and p=0.005 respectively)(Figure). Finally, we performed an ELISA for IFN-alpha on these sorted cells, which was not significantly increased (12 pg/mL) after stimulation with the TLR9 agonist, CpG.These data suggest that reduced GvHD observed in mouse and man after infusion of GM-CSF mobilized stem cell products may be due to both in vivo expansion of Tregs and robust (<1% to 15–20%) mobilization of a novel population of myeloid suppressor cells. SiglecH, a member of the CD33 like immunoglobulin superfamily of lectin proteins, may identify a unique myeloid suppressor cell mobilized by GM-CSF in mice. [Display omitted] Disclosures:Schroeder:Genzyme: Research Funding; Celgene: Research Funding.

Introduction of Differential KIR Activation Mediated Patters of NK Cell Cytotoxicity Against Myeloma

AbstractAbstract 5178 Background:Natural Killer cells (NK cells) are part of the innate immune system. These cells have the ability to recognise and kill malignant cells, like myeloma cells. NK cell activation is tightly regulated by different activating or inhibiting receptors. Killer immunoglobulin like receptors (KIR, CD158) are a family of receptors which have activating as well as inhibitory function. KIR molecules are thought to recognize the HLA-C molecules, which then lead to NK cell signal transduction. Our knowledge about KIR expression and impact on tumor cell control has developed over the last years, but still our understanding of how the receptors are activated in multiple myeloma is limited. We therefore, investigated three different model systems for NK cell alloreactivity: 1) HLA-C/ HLA-C interaction model (KIR-Ligand model), 2) HLA-C/ KIR receptor interaction model, and 3) impact of donor KIR haplotype. Material and Methods:Three different myeloma cell lines (KMS12BM [C1/C1], MOLP8 [C1/C2] and RPMI8266 [C2/C1]) and a NK cell line (NKL) were cultured under standard conditions. NKL cells were transfected with human KIR2DL1 and KIR2DL3 alleles, respectively. For RNAi experiments two siRNA and two control siRNA were used. NK cells from healthy donors were isolated by magnetic end labeling. Enriched NK cells were HLA-typed for expression of HLA-C molecules, KIR receptor expression or KIR haplotype. Thereafter, NK cells were transiently transfected with the siRNA or control siRNA against the KIR2DL1 or KIR2DL3 receptors for up to 48 h. Functional analysis of the NK cell cytotoxicity was measured using a LDH release assay, based on their killing ability against the three fore mentioned myeloma cell lines. Results:Using NK cells that have been HLA-C genotyped as HLA-C1/C1, we observed a rescue of C1/C1 positive meyeloma cell lines in contrast to the C2/C1 myeloma cell line (12% cytotoxicity vs. 38% and 45% cytotoxicity, respectively, p <0.05). KIR receptors, by recognition of HLA-C molecules, are believed to reduce NK cell cytotoxicity. We used NK cells from healthy donors, which express KIR2DL1 (recognizing C2) to evaluate whether receptors' RNAi knockdown could increase the ability of NK cells to lyse target cells. Knockdown of KIR2DL1 in NK cells results in a highly efficient killing of a C2/C2 target cell line (RNAi treated NK cells showed a cytotoxicity up to 70%, p<0.05). To evaluate whether KIR receptors are major inhibitory molecules, we used the NK cell line (wt NKL, a non expressor of KIR receptors) that exhibited high cytotoxic potential against all tested target cell lines (>65% cytotoxicity). This NK cell line was transfected with KIR2DL1 or KIR2DL3 (recognizing C2, or C1). C2 positive myeloma cell lines were significantly rescued when exposed to KIR2DL1 transfected NK cells (wt NKL 72% vs. KIR2DL1 NKL 29%, p <0.05). Similar results were obtained when C1 positive target cell line was co- cultured with KIR2DL3 transfected NK cells. RNAi mediated silencing of KIR overexpression restores protection observed. As last model for the regulation of NK cell cytotoxicity we used KIR haplotyped NK cells and co-cultured them with a C1/C2 myeloma cell line. NK cells with an AA KIR haplotype showed the lowest killing capacity against the myeloma cell line, while BB KIR haplotype had higher killing potential. (46% vs 59%, p=0,005). Conclusions:We have shown that NK cell cytotoxicity is regulated by the HLA-C expression on the target cell as well as the expression of a specific KIR receptor and various KIR haplotypes. All tested models showed significant killing effect on myeloma cells. These data underline the importance of KIR receptor modulation on NK cell alloreactivity against myeloma cells. Disclosures:No relevant conflicts of interest to declare.

Donor KIR-Haplotype B Improved Relapse-Free Survival After Allogeneic Stem Cell Transplantation for Multiple Myeloma

AbstractAbstract 2373NK-cell alloreactivity determined by donor killer-cell immunoglobulin-like receptors (KIRs) has been shown to be predictive for relapse-free survival in myeloid leukemia after allogeneic stem cell transplantation. The role role of NK-cell allo reactivity in patients with multiple myeloma is unclear. Although the ligands for activating KIRs remain elusive, inhibitory KIRs on donor derived NK-cells and HLA class I molecules on the recipient cell determine NK cell alloreactivity. KIR genes on chromosome 19 segregate independently from HLA genes KIR haplotypes can be distinguished in two groups: group A has a fixed number of inhibitory receptors and only one activating receptor (KIR2DS4), while group B haplotype includes more activating receptor genes. Therefore individuals can be categorised as having A-KIR haplotype (A/A), or B-KIR haplotype which contains either A/B heterocygotes or B/B homocygotes KIRs. To analyse the effect of donor KIR-haplotype on outcome in multiple myeloma, we analysed 118 patients with a median age of 51 years (r: 29 – 68) who underwent allogeneic stem cell transplantation. Gender of the study population was predominantly male (n = 75, female: n = 33). Stem cell source was mainly peripheral blood stem cells (n = 105) and derived from unrelated donors (n = 81) or HLA-identical sibling (n = 37). 46 patients received graft from HLA-mismatched donor. The majority of the patients received a melphalan (100 – 140 mg/m2)/fludarabine-based reduced conditioning regimen (N = 106), either as part of an auto-allo-tandem protocol or as an allogeneic salvage protocol after relapse to an autograft. 46 patients had experienced relapse to an autograft prior to allogeneic stem cell transplantation. 79 patients were transplanted from KIR haplotype Bx and 39 from KIR haplotype A. Both groups were well balanced regarding age, HLA match, stem cell source, CMV seropositivity, remission status prior to transplantation, del 13q14, and relapse to prior autograft.After a median follow-up of 5 years the 5-year estimated disease-free and overall survival was better for KIR haplotype B donors in comparison to KIR haplotype A donors (30 % vs. 14 %, p = 0.008, and 49 % vs. 36 %, p = 0.07). The disease-free survival benefit for KIR haplotype B was mainly seen in HLA-matched patients (4y DFS: 39 % vs. 18 %, p = 0.005), while in HLA mismatch transplantation no difference according to KIR haplotype could be detected (26 % vs. 18 %, p = 0.5).The difference in survival in the HLA-matched group was due to higher risk of relapse for KIR haplotype A in the HLA-matched group (cumulative incidence at 1 year 46 % vs. 17 %, p = 0.005).In a multivariate Cox model KIR haplotype A remained a significant factor for worse disease-free (HR: 1.82, p = 0.008) and overall survival (HR: 1.69, p = 0.042). Other significant factors in the multivariate analysis were female donor sex (HR: 1.67, p = 0.04) and chemo-refractory disease at transplantation (HR: 1.76, p = 0.03) for OS and factors for DFS were unrelated donor (HR: 1.83, p = 0.02) and patient's age as continous variable (HR: 1.03, p =0.008) and donor female sex (HR: 1.62, p = 0.03).We conclude that female donor sex and KIR haplotype A are important risk factors for outcome of allogeneic SCT in multiple myeloma and should be considered for donor selection. Disclosures:No relevant conflicts of interest to declare.

Peripheral Blood Stem Cell (PBSC) Haploidentical Transplantation Using a Reduced Intensity Conditioning (RIC), T Cell Replete Approach Proves Both Efficacious and Well Tolerated In a Range of Haematological Malignancies.

AbstractAbstract 4584Haploidentical transplantation is an option for patients who do not have a timely identifiable sibling or volunteer unrelated donor (VUD).Benefits of this stem cell source include donor availability, highly motivated donors and the ability to select the best donor from several relatives taking: age/fitness, cytomegalovirus (CMV) status, ABO group and natural killer cell alloreactivity into account.Historically the high level of human leukocyte antigen (HLA) disparity led to increased graft failure and high rates of acute and chronic graft versus host disease (GVHD). Luznik et al (Blood 2001) demonstrated that the use of post stem cell return cyclophosphamide in RIC haploidentical transplantation (using bone marrow as a stem cell source) reduced acute and chronic GVHD to acceptable levels, but at the expense of higher relapse rates in their cohort.We postulated that the use of PBSC's with their inherently higher T cell complement would reduce relapse rates compared to bone marrow, whilst post cell return cyclophosphamide would reduce acute and chronic GVHD.We present 5 patients treated at our centre using a RIC T cell replete haploidentical transplant protocol utilising PBSC's and post cell return cyclophosphamide.The patients, (median age 51; range 44–58), were treated for: relapsed follicular non Hodgkin's lymphoma (NHL), secondary acute myeloid leukaemia, Mycosis Fungoides and Adult T-Cell Leukaemia/Lymphoma (ATLL).Four patients had received 1st line chemotherapy only and remained chemotherapy sensitive, 3 of whom were in complete remission, one in a partial response. None had undergone a previous transplant. The NHL patient was chemotherapy insensitive following 4 previous lines of chemotherapy, a splenectomy and 2 rejected sibling allografts.Three patients were a major ABO mismatch, the remaining 2 fully matched.Four patients were CMV +/+ and 1 mismatched.HLA disparity ranged from 2–5 alleles (2 and 3 patients respectively).Median CD34+ cell dose returned was 6.98×106 cells/kg (range 4.81–8.00), with a median CD3+ cell dose of 2.36×108 cells/kg (range 1.19–2.97).The conditioning regime used was that of Luznik et al's (Blood 2001) phase I trial:Fludarabine 30mg/m2 day -6 to -2, cyclophosphamide 14.5mg/kg day -6 to -5, total body irradiation 2 Gray day -1, post stem cell cyclophosphamide 50mg/kg day +3 to +4, tacrolimus 1mg IV day +5 onwards, mycophenolate mofetil 15mg/kg TDS day +5 to +35. Outcomes:Four of 5 (80%) patients were fully donor chimeric by day 28 however graft failure with autologous reconstitution due to previously undetected HLA antibodies occurred in 1 patient. This patient reconstituted autologous neutrophils and platelets at 15 and 26 days respectively.Median time to neutrophil and platelet engraftment was 16.5 days (range 14–17) and 12 days (range 11–14) respectively.All 5 patients reactivated CMV (the latest at day 112). With pre-emptive treatment however none developed CMV disease. The incidence of acute GVHD grade II – IV and grade III - IV by day 100 was 40% and 20% respectively. Limited chronic GVHD was seen in 3 patients. 2 were assessed as grade I-II and 1 patient grade III. All cases of acute and chronic GVHD were steroid responsive.In both ATLL patients a sustained suppression of human T-lymphotropic virus (HTLV) viral loads was observed post transplant.One patient subsequently died of sepsis at day 113, the patient who had rejected their graft went on to relapse.The remaining 3 patients continue in CR, performance status 0, currently at day 245, 280 and 438.This data shows that RIC T cell replete haploidentical transplantation using PBSC's is well tolerated and enables both early engraftment and full donor chimerism. The rates of acute and chronic GVHD (40 and 60%) are comparable to sibling and fully matched unrelated donors.All of which has resulted in 60% of patients remaining in CR, including both ATLL patients who have gone on to fully suppress their HTLV viral loads. Disclosures:No relevant conflicts of interest to declare.

Increase In NK Cell Lysis of Leukemic Blasts Due to Loss of Mismatched HLA Haplotype After Haplo-Identical Stem Cell Transplantation

AbstractAbstract 2540 Introduction:Hematopoietic stem cell transplantation (HSCT) from HLA haplo-identical family donors is promising as a therapy for patients with leukemia who are at high risk for relapse. The lower relapse rates and improved survival, especially for patients with acute myelogenous leukemia (AML) who have received HSCT from killer cell immunoglobulin-like receptor (KIR) ligand-mismatched donors, suggest that donor NK cells that are alloreactive against the recipient's cells cause graft-versus-leukemia effects. An Italian group and we identified genomic loss of the patient-specific HLA haplotype in leukemic cells after haplo-identical HSCT. Analysis using SNP arrays revealed that the HLA loss in 29 to 66% of relapsed patients after haplo-identical HSCT was caused by segmental uniparental disomy (UPD) of the HLA region on chromosome 6. This suggested that leukemic cells often escape immunosurveillance through the loss of the mismatched HLA haplotype via the UDP mechanism after haplo-identical HSCT (Vago et al. N Engl J Med. 2009, Villalobos, IB et al. Blood 2010). Since NK cell effector function is tightly regulated by inhibitory KIRs on NK cells that bind to MHC class I on target cells, the escape of leukemic cells from immune surveillance by losing a mismatched HLA antigen might enhance the cytotoxicity of NK cells towards target cells. We examined alterations in donor-derived alloreactive NK cell activity against leukemic blasts of AML patients who relapsed after HLA haplo-identical HSCT. Patients and methods:We enrolled three patients with AML, aged 2, 3 and 12 years, who relapsed after HLA haplo-identical HSCT with T cell depletion of rabbit ATG in vivo. Two patients had AML M7 and the other had M0. Only one donor was KIR ligand-mismatched. Engraftment was achieved in all three patients within 28 days. Relapse occurred 35, 372 and 445 days after HSCT. Mononuclear cells were obtained from both donors and patients before and after HSCT. The NK cells were purified using NK cell selection kits (DYNAL) and measured by conventional 51Cr release assays of leukemic blasts from the patients and the control cell line K562. The mismatched HLA expression between patients and donors on hematopoietic cells was monitored by flow cytometry using anti-HLA antibody (One Lambda). Results:The mean of NK cell killing of the patients' leukemic cells at the same effector target ratio of 10 was significantly higher in donors (50.3%) than in relapsed patients (9.7%). Monitoring of alloreactive NK cell activity after HSCT showed that donor-derived NK cell killing against the patients' leukemic cells gradually decreased in the KIR ligand-mismatched setting. Relapse was minimal but obvious according to HLA monitoring of hematopoietic cells on day 98, which was 7 days after alloreactive NK cell activity was diminished in the patients. Monitoring HLA expression after HSCT also revealed that one patient had leukemic blasts at relapse with loss of the patient's specific HLA haplotype caused by UDP of the HLA region on chromosome 6. Notably, primary leukemic blasts in this patient were not killed by the donor NK cells (2.3%), but leukemic blasts at relapse were efficiently killed (69.0%) after HLA loss of leukemic cells. On the other hand, killing of leukemic blasts at relapse by the patient's NK cells after transplantation was much less effective (19.0%) than that by donor NK cells, although they originated from the same donor (Figure). Because haplotype loss of HLA caused by UDP does not change the status of the KIR ligand in patients with homozygous HLA-Cw, we further examined the expression of ULBP1-3 that are ligands for the activating NK receptor, NKG2D, on leukemic blasts before and after HLA loss. We found upregulated ULBP-2 expression on leukemic cells after the loss of HLA. Conclusions:Donor NK cells efficiently killed patients' AML blasts at relapse but NK cell activity in patients against their own leukemic blasts was impaired after HSCT. These findings indicate a rationale for donor NK cell infusions after HLA haplo-identical HSCT to avoid decreasing NK cell alloreactivity and to prevent the escape of leukemic cells from allo-immune surveillance by donor cytotoxic T lymphocytes. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinical Grade Isolation of Regulatory T Cells From G-CSF Mobilized PBSC Improves with Initial Depletion of Monocytes.

AbstractAbstract 1176We have recently demonstrated that G-CSF mobilized peripheral blood stem cells (PBSC) CD4+CD25+FoxP3+ cells (Tregs) prevent anti-CD34+ hematopoietic stem cells T cell alloreactivity in-vitro and co-transplantation of CD34+ cells and Tregs does not affect human stem cell engraftment in NOD/SCID mice (Mahmud D et al. Biol Blood Marrow Transplant, 2010). Since only a small number of Tregs can be isolated from normal peripheral blood we examined whether PBSC can be a useful source of Tregs for future clinical trials. Five leukapheresis products from healthy donors who received rh G-CSF at 10 ug/kg daily for 5 days were processed using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA). CliniMACS CD34 reagent was initially used to isolate CD34+ cells. To isolate Tregs a 2-step procedure was initially utilized. A cocktail of clinical grade CD14, CD8 and CD19 reagents was mixed with the CD34- cells and depletion of monocytes, cytotoxic T cells and B cells was obtained by using the Depletion 2.1 program. The CD4+ cells were then enriched in Tregs by positive selection of CD25+ cells using a clinical grade CD25 reagent (Miltenyi). Because PBSC contain large amount of myeloid cells, and particularly monocytes, this clinical scale 2-step strategy was compared with a 3-step method that included an initial negative selection of CD14+ monocytes, followed by negative selection of CD8+ and CD19+ cells and a positive selection of CD25+ cells. Prior to isolation, the average proportion of CD4+CD25+ cells in PBSC was 0.77±0.26% in 5 separate PBSC products. After the 2-step process the proportion of CD4+CD25+ cells was 35±33% (n=3) vs 72±1% after the 3-step process. Therefore, utilizing the 3-step approach a better yield of Tregs was observed (10 vs 60%). Intracellular expression of FoxP3 was on average 74% in CD4+CD25+ cells obtained with a 3-step process. Contamination of different cell subsets in the final products enriched in Tregs was largely superior following the 2-step as compared to 3-step isolation method. Contaminating monocytes were, on average, 43 vs 5.7%, and contaminating CD8 and CD19+ cells were 12 vs 1.7% and 0.9 vs 0.3%, respectively. In the two procedures using a 3-step approach the final absolute number of Tregs isolated from products containing on average 30 × 109 mononuclear cells, was 95 and 93 × 106, respectively. These findings obtained using clinically available reagents and device, suggest that depletion of monocytes may improve the purity of Treg cell population isolated from PBSC. PBSC may represent a valuable source of Tregs for future clinical trials. Disclosures:No relevant conflicts of interest to declare.

TIM-3: A Novel Regulatory Molecule of Alloimmune Activation

T cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4(+)FoxP3(-) and CD8(+) T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4(+) T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8(+) cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4(+) T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4(+) T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response.

Alloreactive natural killer cells promote haploidentical hematopoietic stem cell transplantation by expansion of recipient-derived CD4+CD25+ regulatory T cells

Alloreactive NK cells (Allo-NKs) have been shown to exert advantageous effects on the outcomes of haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) for cancer treatment. However, the mechanisms of action of Allo-NKs remain unclear. We established a novel Haplo-HSCT conditioning regimen composed of Allo-NKs and a low dose of immunosuppressive drugs (Allo-NKs + Chemo) to investigate alternative mechanisms besides direct cytotoxicity. The inhibitory effects of different cell subsets on the donor-recipient mixed lymphocyte reactions (MLRs) were evaluated after Haplo-HSCT. The quantities and functions of CD4(+) CD25(+) regulatory T cells (Tregs) and dendritic cells (DCs) in the spleen and the thymus were examined. Our results showed that the Allo-NKs + Chemo regimen induced systemic tolerance, and that CD4(+) CD25(+) Tregs played a significant role in inducing and maintaining systemic tolerance after Haplo-HSCT. Alloreactive NK cells promoted the expansion of recipient-derived CD4(+) CD25(+) CD127(-) Tregs in the thymus and the spleen which could be amplified in vitro by the immature donor-derived DC subset isolated from the thymus of Allo-NKs + Chemo-treated mice. Our findings suggested that Allo-NKs are capable of inducing systemic tolerance after Haplo-HSCT by assembling donor-derived immature DCs to expand recipient-derived Treg cells in the thymus.

An MHC-Defined Primate Model Reveals Significant Rejection of Bone Marrow After Mixed Chimerism Induction Despite Full MHC Matching

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC-defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade-/sirolimus-mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan-based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient-rather than donor-derived. Surprisingly, even in MHC-matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen-experienced T cells, and transplant rejection was associated with the acquisition of donor-directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the post-immunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen-experienced phenotype, and ultimately, to transplant rejection.

Natural killer cells and tumor control

After hematopoietic cell transplantation (HCT) donor-derived natural killer (NK) cells kill tumor cells to prevent relapse and mediate other beneficial clinical effects including control of infections without inducing graft-vs.-host disease (GVHD). Understanding the determinants of NK cell alloreactivity and function will support improvements in the design of HCT and adoptive cellular therapies. Refinements to the model of NK cell education or licensing have been made which will inform strategies to develop functional alloreactive NK cells for therapeutic use. Differences in NK cell function have been shown to be dependent on the nature of the stimuli. Recent advances have been made in our understanding of the role of activating NK receptors on education and outcome after HCT. The use of adoptively transferred NK cells to treat hematopoietic malignancies has been expanding. New approaches to modulate target sensitivity to NK cell-mediated killing are under development. NK cells play an important role in the therapeutic efficacy of HCT, with effects on control of infections, GVHD, engraftment and relapse prevention. Recent advances in our understanding of NK cell biology will support improvements in our ability to exploit NK cells to treat cancer.

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use. We describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells. NK cells (6.0 ± 1.2 x 10(8)) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR+ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML. The approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.

Interleukin-17 Promotes Early Allograft Inflammation

Acute cellular rejection of organ transplants is executed by donor-reactive T cells, which are dominated by interferon-gamma-producing cells. As interferon-gamma is dispensable for graft destruction, we evaluated the contribution of interleukin-17A (IL-17) to intragraft inflammation in major histocompatibility complex-mismatched heart transplants. A/J (H-2(a)) cardiac allografts placed into wild-type BALB/c (H-2(d)) mice induced intragraft IL-17 production on day 2 after transplant. Allografts placed into BALB/c IL-17(-/-) recipients demonstrated diminished production of the chemokines CXCL1 and CXCL2 and delayed neutrophil and T cell recruitment. However, by day 7 after transplant, allografts from IL-17(-/-) and wild-type recipients had comparable levels of cellular infiltration. The priming of donor-specific T cells was not affected by the absence of IL-17, and the kinetics of cardiac allograft rejection were similar in wild-type and IL-17(-/-) recipients. In contrast, IL-17(-/-) mice depleted of CD8 T cells rejected A/J allografts in a delayed fashion compared with CD8-depleted wild-type recipients. Although donor-reactive CD4 T cells were efficiently activated in both groups, the infiltration of effector T cells into allografts was impaired in IL-17(-/-) recipients. Our data indicate that locally produced IL-17 amplifies intragraft inflammation early after transplantation and promotes tissue injury by facilitating T cell recruitment into the graft. Targeting the IL-17 signaling network in conjunction with other graft-prolonging therapies may decrease this injury and improve the survival of transplanted organs.