T-cell Depleted Bone Marrow Research Articles

Attenuation of Hepatic Graft-versus-host Disease in Allogeneic Recipients of MyD88-deficient Donor Bone Marrow.

Acute graft-versus-host-disease (GVHD) is characterized by selective damage to the liver, the skin, and the gastrointestinal tract. Following allogeneic hematopoietic stem cell transplantation, donor bone marrow (BM) cells repopulate the immune system of the recipient. We previously demonstrated that the acute intestinal GVHD (iGVHD) mortality rate was higher in MyD88-deficient BM recipients than that in the control BM recipients. In the present study, the role of MyD88 (expressed by donor BM) in the pathophysiology of hepatic GVHD (hGVHD) was examined. Unlike iGVHD, transplantation with MyD88-deficient T-cell depleted (TCD) BM attenuated hGVHD severity and was associated with low infiltration of T cells into the liver of the recipients. Moreover, GVHD hosts, transplanted with MyD88-deficient TCD BM, exhibited markedly reduced expansion of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) in the liver. Adoptive injection of the MDSC from wild type mice, but not MyD88-deficient mice, enhanced hepatic T cell infiltration in the MyD88-deficient TCD BM recipients. Pre-treatment of BM donors with LPS increased MDSC levels in the liver of allogeneic wild type BM recipients. In conclusion, hGVHD and iGVHD may occur through various mechanisms based on the presence of MyD88 in the non-T cell compartment of the allograft.

MyD88 in Donor Bone Marrow Cells Is Critical for Protection from Acute Intestinal Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation is an important therapeutic modality used to treat malignancies of hematopoietic origin, such as leukemia and lymphoma. However, development of graft-versus-host disease (GVHD) causes non-relapse mortality and substantial morbidity of recipients. Myeloid differentiation factor 88 (MyD88), a major adaptor mediating TLR signaling, is also known to deliver pro-inflammatory signals. Activation of inflammatory signaling through MyD88 plays a key role in the expansion of myeloid-derived suppressor cells (MDSC) which are a heterogeneous population of immature myeloid ells with anti-inflammatory activity. To explore the contribution of MyD88 expressed by donor bone marrow (BM) cells to development of GVHD, we induced GVHD using T-cell-depleted BM (TCD-BM) isolated from MyD88-deficient (MyD88KO) mice and T cells isolated from wild-type (WT) mice. We employed C57BL/6 (H-2b) → B6D2F1 (H-2b/d) mouse model of GVHD, which differ at major and minor histocompatibility loci. Lethally irradiated B6D2F1 recipient mice were transplanted with either T cell-depleted bone marrow (TCD BM, 5 x 106) from either WT or MyD88KO mice together with WT spleen T cells (1 x 106). Transplantation with MyD88KO TCD BM aggravated GVHD; serious gut damage was evident, with infiltration of T cells specifically into the intestines of recipients. GVHD hosts transplanted with MyD88KO TCD BM exhibited markedly reduced expansion of MDSC. GVHD aggravation after transplantation with MyD88KO TCD-BM, associated with high-level T cell infiltration into the intestine and insignificant expansion of MDSC, was reproduced in another minor histocompatibility mismatch model (C57BL/6 → BALB.B). We next examined allogeneic T cells in the spleens of GVHD hosts in terms of the expression levels of CCR9, which are known to be associated with T cell migration to the intestinal mucosa and the proportion of CCR9 positive cells in CFSE low CD8+ T cells was higher in recipients of MyD88KO TCD BM than WT controls. In parallel, the levels of CCL25 were more highly expressed in the gut of MyD88KO recipients than WT controls. Mixed leukocyte cultures of CFSE- labeled C57BL/6 T cells and irradiated B6DF1 feeder cells were prepared in the presence of MDSC isolated from MyD88KO or WT mice. T cells, co-incubated with MDSC isolated from MyD88KO BM, exhibited a greater extent of CFSE dilution and less Annexin V staining, compared to T cells co-incubated with cells from WT BM. Moreover, MDSC from recipients of MyD88KO TCD BM exhibited a reduced suppressive function, compared to their WT counterparts. Next, we determined whether insufficient expansion of and ineffective suppression by MDSC caused severe GVHD in recipients of MyD88KO TCD BM. Supplementation of transplanted mice with MDSC from WT mice, not from MyD88KO mice, attenuated the severity of GVHD and reduced intestinal T cell infiltration in recipients of MyD88KO TCD BM. To verify the importance of MyD88-mediated signaling by MDSC in protection against severe GVHD, we determined if transplantation with TCD-BM cells containing high levels of MDSC attenuated the severity of GVHD. Pre-treatment of BM donors with lipopolysaccharide increased the frequencies of MDSC and the amounts of MyD88 transcripts in TCD-BM transplant, and alleviated the severity of GVHD and intestinal T-cell infiltration. To explore whether MDSC expansion levels could be used to predict the severity of intestinal GVHD, the T/MDSC ratios were calculated in blood of patients at the time of engraftment and were significantly higher in patients with intestinal GVHD ≥ grade 2. In conclusion, we have shown that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal acute intestinal GVHD. DisclosuresNo relevant conflicts of interest to declare.

Intestinal Helminth Colonization Regulates Lethal Graft Versus Host Disease and Preserves Graft Versus Tumor in Bone Marrow Transplanted Mice

Introduction.Graft versus host disease (GVHD) is a frequent and severe complication of allogeneic bone marrow transplantation (BMT). Novel and less toxic treatment regimens are needed to suppress GVHD and preserve graft versus tumor (GVT). Self-limited intestinal helminth colonization stimulates host regulatory T cell (Treg) subsets. The intestine is a prime organ in GVHD generation, with Tregs playing a pivotal role in controlling GVHD. Treg-mediated suppression of GVHD preserves GVT. We hypothesized that self-limited intestinal colonization with helminths protects BMT mice from lethal acute GVHD and preserves the GVT.Methods. Three weeks after administering 3rd stage adult Heligmosomoides polygyrus larvae into 5-6 week old male WT Balb/C recipient mice (H2d), we initiated H2b→H2d MHC I/II mismatch model of acute GVHD by total body irradiation (TBI; 850 cGy) and administration of total splenic T cells and T cell depleted (TCD) bone marrow (BM) cells from male C57BL/6 WT (MHC: H2b) donors into uninfected control and helminth-infected male wild-type (WT) Balb/C (MHC: H2d) bone marrow recipients. In certain experiments, we used donor T cells from TGFβ RII dominant negative (DN) (TGFβ RII DN) mice (MHC: H2b) whose T cells are unresponsive to TGFβ-mediated immune regulation due to over-expression of a truncated TGFβ receptor II. Th1 (IFNγ, TNFα) cytokine generation was analyzed by ELISA. Splenic and mesenteric lymph node (MLN) cells were stained for H2b, H2d, T cell surface markers and FoxP3. Tissues were analyzed for GVHD-related inflammation. GVT was assessed in uninfected and helminth-infected BMT recipients by administration of luciferase expressing A20 leukemia/lymphoma cells (A20-luc; H2d) and IVIS imaging. Statistical and survival difference between groups was determined by Student’s t-test and Kaplan Meier curves, respectively.Results. Helminths increased ~3-fold CD4 T cell and FoxP3+ Treg TGFβ generation (p<0.01 btw uninfected and helminth-infected). Helminths suppressed donor T cell Th1 cytokine production (p<0.05 btw control and helminth-infected mice (N=3)) and regulated clinical as well as histopathological (colon and lung) GVHD score (p<0.05 between control and helminth-infected, for each parameter from multiple experiments). Helminths promoted the survival of recipient T cells after TBI (Spleen control: 1.4±0.9 x 104 vs. helminth-infected: 11.0±6.9 x 104 CD3+ T cells (N=8; p<0.05); MLN control: 0.09±0.09 x 104 vs. helminth-infected: 6.0±5.2 x 104 (N=8; p<0.001)). Surviving T cells in H. polygyrus-colonized mice were enriched for FoxP3+ Tregs (spleen: 1.16±0.6 x 104 and MLN: 0.65 ±0.56 x 104 FoxP3+ Tregs). Recipient T cells enriched for Tregs survived during GVHD, as analyzed 6 days after BMT (3.3±1.1% of splenic and 15.9±3.5% of MLN host T cells being FoxP3+ Tregs). The number of recipient T cells in uninfected BMT mice was low for further Treg analysis. Helminths increased the number of GVHD-regulating donor-derived H2b+ FoxP3+ Tregs (Spleen: 66±32 x 103 (control) vs. 206±73 x 103 (helminth-infected) Tregs per mouse (p<0.05; N=6); MLN: 1.6 ±1.1 x 103 (control) vs. 4.9±2.0 x 103(helminth-infected) Tregs per mouse (p<0.05; N=4). Helminth infection was associated with >15 weeks survival of 40% of recipients, while all uninfected BMT recipient mice died of GVHD (p<0.01 between uninfected (N=10) and helminth-infected (N=10) WT recipients). Helminths did not promote the survival of recipients, when T cells unresponsive to TGFβ were used as donor lymphocytes. Helminths preserved the GVT in BMT mice transferred A20-luc. All uninfected and helminth-infected TCD BM transferred recipients died within few weeks displaying significant luciferase activity, while no luciferase signal was evident at day 70 in surviving (5/9) helminth-infected mice administered TCD BM and splenic T cells along with A20-luc. Uninfected mice (9/9) transferred splenic T cells, TCD BM and A20-luc died of GVHD without showing luciferase activity.Conclusions. Our results show that (1) helminths stimulate TGFβ producing host T cells and FoxP3+ Tregs that survive BMT conditioning with TBI; (2) helminths regulate GVHD by inducing donor Tregs and in a TGFβ dependent manner; (3) helminths preserve GVT. With a safe side-effect profile, helminths or helminth products have the potential to be investigated as an alternative strategy to prevent or treat GVHD. DisclosuresNo relevant conflicts of interest to declare.

CD4+FoxP3+ Regulatory T Cells Regulate B Cell Differentiation and Induce Tolerance to Bone Marrow Grafts

CD4+FoxP3+ regulatory T cells (Treg) are a rare cell population that is responsible for peripheral immune tolerance. Treg adoptive transfer has proved to be an effective treatment for graft versus host disease prevention in several preclinical and clinical studies. The impact of Treg on immune reconstitution and bone marrow engraftment after transplantation has been less well studied.We treated C57BL/6 FoxP3-DTR mice that carry the diphtheria toxin (DT) receptor in the promoter of the FoxP3 gene with DT resulting in a complete ablation of Treg (percentage of FoxP3+CD4+ cells over CD4+ cells < 0.2% in peripheral blood, spleen, lymph nodes, thymus and bone marrow). Transplantation of lethally irradiated (TBI 10 Gy) Treg depleted mice with allogeneic (BALB/C or FVB/N) T-cell depleted bone marrow (TCD BM) resulted in rejection or reduced donor chimerism (p < 0.01). Treg depletion favored host CD4+ (p < 0.001), CD8+ (p < 0.01) and GR1+ cell persistence (p < 0.01) and delayed B cell reconstitution (p < 0.001). Adoptive transfer of purified host type Treg in vitro activated with Interleukin-2 (IL-2) and anti-CD3/CD28 beads to DT treated mice rescued engraftment (p < 0.01) and boosted B cell reconstitution (DT treated mice that received Treg vs DT treated mice only p < 0.001; DT treated mice that received Treg vs untreated mice p < 0.01). Moreover Treg ablation resulted in rejection or reduced donor chimerism (p < 0.01) in mice transplanted with allogeneic (FVB/N) purified Lin-Sca1+cKit+ hematopoietic stem cells (HSCs) demonstrating that Treg promote donor HSC engraftment without interacting with donor derived facilitating cells.We explored the mechanism through which Treg impact donor engraftment and immune reconstitution and observed that Treg depleted mice transplanted with syngeneic (C57BL/6 CD45.1) TCD BM engrafted (p > 0.05) but had markedly delayed B cell reconstitution (p < 0.01) thus Treg promote donor B cell differentiation in manner not dependent upon alloreactivity. FACS analysis of bone marrow cells of syngeneic transplanted mice showed higher numbers of donor Lin-Sca1+cKit+ HSCs (p < 0.05) and donor Lin-Sca1+cKit+Flt3+ lymphoid progenitors (p < 0.05) while numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.01) were reduced after Treg depletion demonstrating a block of maturation in the early phases of B cell differentiation. Confocal microscopic analysis of femurs after transplantation with TCD BM were used to determine the spatial relationship between Treg, B cells and HSCs. Treg localize near the endosteum (p < 0.05) and cluster in the epiphyseal areas where donor HSCs and B220+ B cells were mainly detectable suggesting that Treg act as an immunological barrier for HSCs and B cell progenitors, providing a protective immunological niche. Further, adoptive transfer of IL-2 and CD3/CD28 bead activated Treg induced B cell reconstitution in non irradiated immune deficient BALB/C rag2-/-γc-/- mice that received an infusion of allogeneic (C57BL/6) TCD BM. Bone marrow analysis of these mice revealed higher numbers of B220+IgM-CD19+cKit+ Pro-B cells (p < 0.05) and total CD19+ cells (p < 0.05) in mice that received Treg. Analysis of the sera after Treg adoptive transfer showed reduced production of inflammatory cytokines and chemokines such as Interferon-γ, Interleukin-12, Interleukin-13, Interleukin-1b, CXCR-9 and CXCR-10 and increased production of VEGF.In conclusion, our findings clearly indicate that Treg act as a key regulator of B cell differentiation promoting production of mature B cells. This effect is not dependent on alloantigen recognition and Treg localization after transplantation suggests that Treg play a role in building the donor HSC and B cell precursor niche. Finally, adoptive transfer of Treg enhances B cell immune reconstitution and induces tolerance to allogeneic bone marrow grafts even in the absence of conditioning providing a new tool for clinical translation in children with severe combined immune deficiencies or hemoglobinopathies and in patients undergoing organ transplantation. DisclosuresNo relevant conflicts of interest to declare.

A Radio-Resistant Perforin-Expressing Lymphoid Population Controls Allogeneic T Cell Engraftment, Activation and Onset of Gvhd

IntroductionImmunosuppressive pre-transplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplant (BMT). The role of residual post-conditioning recipient immunity in determining engraftment is poorly understood. Although recipient perforin has previously been shown to modulate myeloid engraftment from mouse bone marrow, and adoptive transfer of natural killer (NK) cells can ameliorate the onset of graft-versus host disease (GVHD), the role of perforin-expressing endogenous recipient NK and NKT cells in modulating donor T cell engraftment has not been described. Using an MHC-mismatched mouse model, we examined the role of perforin-expressing NK cells in regulating both the rate and activation status of donor lymphocyte engraftment after allogeneic transplantation.MethodsAn MHC-disparate model of BMT was established transplanting BALB/c donor BM (H-2Kd), into female 6-14 week old C57BL/6 WT and C57BL/6.perforin KO (H-2Kb) recipients. On day 0, recipient mice were administered a split dose of lethal radiation using a caesium source (2 x 6 gray), and injected i.v. with 5e6 T cell depleted BM (TCD-BM) cells from MHC-mismatched or syngeneic donors. On day 2, recipient mice were injected i.v. with 1e6 BALB/c purified splenic T cells at a 2:1 CD4+:CD8+ T cell ratio. Mice were monitored daily, and killed at selected time points post-transplant (typically day 5-7 for short term engraftment, or day 20 for long term engraftment) to examine donor lymphoid cell engraftment.ResultsAn HLA-mismatched BMT mouse model demonstrated that both the rate and proportion of donor lymphoid cell engraftment, and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5-7 days post BMT in perforin-deficient (pfn-/-) recipients, compared with wild-type (WT). Critically, we found that the absence of perforin resulted in the increased production of pro-inflammatory cytokines, in particular IL-6, from engrafting donor T cells. IL-6 has recently been identified as a primary driver of both mouse model and clinical GVHD. Correlating with pro-inflammatory cytokine secretion, effector memory donor T lymphocytes expanded more rapidly in pfn-/- recipients than in WT mice, possibly arising from more rapid proliferation or selective survival of these donor cells. In WT recipients, donor lymphoid cell engraftment was enhanced to that seen in pfn-/- recipients, by depleting NK cells prior to BMT, demonstrating that a perforin-dependent, NK-mediated host-versus graft effect limits the rate of donor cell engraftment and T cell activation. We found that radiation-resistant NKT cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the host-versus graft effect. Furthermore, reduced pre-transplant irradiation doses in pfn-/- recipients permitted long-term donor lymphoid cell engraftment and improved survival (Figure 1).Figure 1: Reduced total body irradiation in perforin-deficient mice permits rapid donor lymphoid cell engraftment. On day 0, WT or pfn-/- (KO) mice were irradiated with variable doses (12-6 gray), and injected i.v. with 5e6 TCD-BM cells from BALB/c donors. On day 2, recipient mice were injected i.v. with 1e6 splenic BALB/c T cells. On day 7 (A) and day 20 (B) after BMT, the BM cells of WT or KO mice were stained for H-2Kd (donor cells) and engraftment determined as a percentage of donor lymphocytes. (C) Survival of KO mice administered 9 gray (closed circles), 7.5 gray (triangles) and 6 gray (lines) after BMT described above. [Display omitted] [Display omitted] [Display omitted] ConclusionsThe challenge in clinical HSCT is to promote reliable donor engraftment and full immunological reconstitution whilst minimizing pre-transplant conditioning and associated toxicity. Our findings suggest that suppression of perforin activity or selective depletion of recipient NK cells prior to BMT may allow the combined advantage of reduced transplant toxicity whilst maintaining donor engraftment and promoting the graft versus tumour effect. DisclosuresNo relevant conflicts of interest to declare.

CD19 CAR T Cells Maintain Efficacy in the Allogeneic Environment but Mediate Acute Graft-Versus-Host-Disease Only in the Presence CD19+ Acute Lymphoblastic Leukemia

A significant portion of patients with relapsed acute lymphoblastic leukemia (ALL) who are eligible for treatment with chimeric-antigen-receptor (CAR) T cells have undergone a previous hematopoietic stem cell transplantation. We and others have previously demonstrated that the allogeneic environment, even in the presence mild graft-versus-host disease (GVHD), severely impairs tumor-directed T-cell immunity. To study the behavior of CAR cells in the allogeneic setting we chose a murine model of minor mismatched allogeneic T-cell depleted bone marrow transplantation (BMT) followed by CAR T (CD19-28-z) cell infusion in recipients bearing a murine B-precursor ALL model (positive for CD19). Importantly, the leukemia persists following myeloablative radiation thus mimicking post-transplant minimal residual disease. Donor-derived, post-transplant injection of CD19 CAR T cells eliminated residual leukemia in the syngeneic as well as the allogeneic settings. CD19 CAR T cells harvested from allogeneic recipients maintained in-vitro ability to produce IFN-gamma and degranulate in the presence of ALL ex vivo, at levels comparable to syngeneic CD19 CAR T cells. However, CAR T cells administered in the allogeneic environment had the potential to mediate severe acute GVHD with early mortality of recipients not typically seen in this minor mismatch model. This occurred across multiple T cell doses capable of clearing leukemia (0.3e6-5e6), in transduced CD19 CAR T cells generated from donors tolerized to allogeneic antigens, and when CD19 CAR T cell infusion was delayed following BMT. In-vivo tracking of transferred cells showed comparable expansion and persistence of the CD19 CAR T cells in the allogeneic and syngeneic environments. However, syngeneic CAR T cells tended to develop later into central memory T cells (CD62L+CD44+), whereas the profile of allogeneic cells was significantly skewed toward effector T cells (CD62L-CD44+). Remarkably, the process of CAR-driven acute GVHD in this minor mismatch model was only seen in the presence of leukemia, indicating a CAR-target interaction influences the induction of GVHD. Indeed, pro-inflammatory cytokines IFN-gamma and IL-6 were elevated only in the presence of both ALL and CAR T cells, whereas TNF alpha levels were undetectable in all instances. We are currently testing whether neutralization of cytokines can prevent GVHD in these models. Altogether, these data demonstrate efficacy of CD19 CAR to clear residual leukemia in an immunocompetent mouse model, and maintain initial cytotoxicity despite the potentially suppressive allogeneic environment. However, we demonstrated potential risks of allogeneic CAR T cells aggravating GVHD in the presence of residual leukemia, likely via cytokine-mediated manner. Clinically, as IL-6 was shown to be significant in cytokine release syndrome, this may represent a murine model to study potential interventions. DisclosuresNo relevant conflicts of interest to declare.

Chimerism Induction Without GVHD Following Low Intensity Conditioning In MHC Disparate Recipients: Combining The Benefit Of Cyclophosphamide Post Transplant With The Tolerogenicity Of Megadose T Cell Depleted HSCT

Achieving allogeneic T-cell depleted BM (TDBM) engraftment under non-myeloablative conditioning represents a potentially safe and attractive approach for generating tolerance as a platform for organ transplantation and for the treatment of non-malignant diseases. However, overcoming the abundant host anti-donor T-cells (HADTC) surviving such protocols remains a major challenge.The potential role of megadose T cell depleted BM transplants in overcoming this barrier was shown when using 6.0Gy sublethal TBI, but chimerism could not be attained upon further reduction of the conditioning. An alternative approach currently in clinical use is based on the administration of high dose cyclophosphamide at days 3-4 after transplantation. However, attempts have been limited to T cell replete transplants containing at least 2x106 T cells/Kg. The latter are clearly critical for achieving engraftment of mis-matched transplants but they are also associated with a significant risk for GVHD.In the present study, we evaluated the potential benefit of combining these two approaches. Initially, regular (5x106) or high dose (25x106) Balb/c-Nude BM cells (fully depleted of T cells by virtue of the 'nude' phenotype) were transplanted (day 0) into fully allogeneic recipients (C3H/Hen). Conditioning included T cell debulking (TCD) with anti-CD4 and anti-CD8 antibodies (300µg each) delivered on day -6, and exposure to 2.0 Gy total body irradiation (TBI) on day -1. High dose Cyclophosphamide (CY) (100mg/kg) was administered on days +3 and +4. Chimerism analysis on days 35, 95, 180 and 225 revealed that none of the BM recipients that were transplanted with a regular dose of 5x106 T depleted BM in the presence or absence of CY, expressed donor type chimerism. In contrast , 4/7 mice receiving 25x106 cells and CY, exhibited (on day 225) more than 47.7±9. % multi-lineage chimerism. The stability of chimerism over more than 7 months indicated that tolerance was likely achieved. Indeed, this was confirmed by subsequent acceptance of donor type Balb/c skin grafts. Thus, 3/4 chimeric mice accepted Balb/c skin and rejected 3rd party C57BL/6 skin, while all mice that were inoculated with regular dose of BM cells in conjunction with CY (7/7), rejected both donor and 3rd party grafts.Encouraged by these results, we attempted to determine the optimal CY and radiation dose, to improve chimerism induction. Thus, mice receiving increasing CY dose of 125 or 150 mg /kg did not exhibit significant enhancement of chimerism. In addition, as shown in Fig1, while most of the recipients that were irradiated with 2Gy (13/15), 2.5Gy (6/6), 3Gy (4/5), or 3.5Gy, TBI (5/5) did not differ significantly in chimerism level (average= 69.1±24.7% at 300 days post transplant), further reduction of irradiation dose to 1.0Gy TBI, resulted in significantly reduced chimerism (average=25.4±26.2%, p≤0.003 ). Importantly, acceptance of donor type skin graft was further manifested when tested in chimeric mice conditioned with 2GY (9/13), while 3rd party skin was completely rejected (Fig.1). [Display omitted] Considering previous suggestions by Ildstad et al. that a subset of CD8+ TCR- BM cells is critical for achieving donor type chimerism, we next determined whether such cells are indeed critical for engraftment of megadose T cell depleted BM transplants. To that end, CD8+ cells were depleted from the Balb/c -'nude' mega dose BM preparation, and chimerism induction was compared to control non-depleted 'nude' BM cells. The results of two independent experiments revealed no significant difference (P≥0.9974) in chimerism levels between mice transplanted in the presence (47.3±31, n=16) or absence (47.3±29, n=15) of CD8+TCR- cells, suggesting that such cells are not essential when using megadose T cell depleted transplants in conjunction with CY.Taken together, our results demonstrate that combination of T cell depleted megadose hematopoietic stem cells with CY administration post transplantation can lead to marked chimerism following low intensity conditioning, in contrast to using each approach alone. Importantly, there is no need to preserve CD8+TCR- cells in the transplant preparation. Such transplants, which are completely free of GVHD risk, could potentially pave the way for a safer tolerance induction modality in organ transplantation and in other non-malignant indications in which the risk associated with GVHD is not justified. Disclosures:Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Central Memory T Cells That Express High Level Of CD44 Mediate Strong Anti-Tumor Response Without Gvhd

Several groups have demonstrated directly and indirectly that central memory T cells (TCM) have decreased ability to induce graft-versus-host disease (GVHD) compared with naïve T cells (TN). We have previously demonstrated that TCM expressing high level of CD44 (CD44high TCM) do not cause GVHD. In this study, we further defined the role of CD4+ and CD8+ subsets of this population in GVHD using the same GVHD model as we used previously (C57BL/6→BALB/c). While all CD4+ TN recipients developed lethal GVHD and died within 28 days after transplantation, none of the recipients of CD4+CD44high TCM developed GVHD and all survived more than 100 days after transplantation (P<0.01). Similarly, 36% of CD8+ TN recipients developed lethal GVHD but none of CD8+CD44high TCM recipients developed GVHD (P<0.05). One of the unique features of effector memory T cells (TEM) is that these cells are able to facilitate stem cell engraftment and mediate some degree of anti-tumor response without causing GVHD. To test the ability of TCM to deplete host radioresistant T cells, BALB/c recipients were lethally irradiated (8.5 Gy) and transplanted with 1x107 T-cell-depleted (TCD) bone marrow cells together with graded numbers of T cell subsets from C57BL/6. Host T cells persist in all mice receiving only TCD bone marrow cells. Addition of 1x105 of either TN or CD44high TCM to the graft led to 100% depletion of all host radioresistant T cells. In contrast, none of 1x105 TEM recipients and only 50% of 5x105 TEM recipients depleted all host radioresistant T cells. These data indicate that TN and CD44high TCM have equivalent abilities to deplete host radioresistant T cells and both of them are at least 5 times more potent than TEM. To test the ability of CD44high TCM to mediate anti-tumor effects, host-type leukemia/lymphoma cells (BCL1) cells were added to the stem cell graft. All recipients transplanted with TCD bone marrow together with BCL1 cells developed tumor and died within 28 days after transplantation. Addition of 1x104 TN and CD44high TCM protected 42% and 36% of the recipients from developing tumor respectively. In contrast, only 7% of TEM recipients were protected (P<0.05) at the same dose level. Similarly, addition of 1x105 TN and CD44high TCM protected 79% and 80% of the recipients from developing tumor respectively. However, only 25% of 1x105 TEM recipients were protected (P<0.01). These data indicate that TN and CD44high TCM have equal anti-tumor activity and both of them are at least 10 times more potent than TEM. Similar results were obtained in CD8 subset of CD44high TCM. A critical question is why CD44high TCM are able to kill host radioresistant T cells and tumor cells so efficiently without inducing GVHD. Even though CD44high TCM were able to respond to alloantigens, bioluminescence imaging analyses suggest that CD44high TCM also mediate an abortive alloresponses upon transfer into allogeneic recipients. Using an in vivo functional assay, we further demonstrated that alloreactive CD44high TCM lost the reactivity over time, suggesting a mechanism by which CD44high TCM were able to eliminate host resistance without causing GVHD. In conclusion, these data demonstrate that selective transplantation of CD44high TCM is able to efficiently separate anti-tumor effects from GVHD. Disclosures:No relevant conflicts of interest to declare.

Effects of easy-to-perform procedures to reduce bacterial colonization withStreptococcus mutansandStaphylococcus aureuson toothbrushes

It is well known that dental caries and periodontitis are the consequence of bacterial colonization and biofilm formation on the enamel surface. The continuous presence of bacterial biofilms on the tooth surface results in demineralization of the tooth enamel and induces an inflammatory reaction of the surrounding gums (gingivitis). The retention and survival of microorganisms on toothbrushes pose a threat of recontamination especially for certain patients at risk for systemic infections originating from the oral cavity, e.g., after T-cell depleted bone marrow transplantation. Thus, the effects of different decolonization schemes on bacterial colonization of toothbrushes were analyzed, in order to demonstrate their applicability to reduce the likelihood of (auto-)reinfections. Toothbrushes were intentionally contaminated with standardized suspensions of Streptococcus mutans or Staphylococcus aureus. Afterwards, the toothbrushes were exposed to rinsing under distilled water, rinsing and drying for 24 h, 0.2% chlorhexidine-based decolonization, or ultraviolet (UV) radiation. The remaining colony forming units were compared with freshly contaminated positive controls. Each experiment was nine-fold repeated. Bi-factorial variance analysis was performed; significance was accepted at P < 0.05. All tested procedures led to a significant reduction of bacteral colonization irrespective of the toothbrush model, the brush head type, or the acitivity state. Chlorhexidine-based decolonization was shown to be superior to rinsing and slightly superior to rinsing and drying for 24 h, while UV radiation was similarly effective as chlorhexidine. UV radiation was slightly less prone to species-dependent limitations of its decolonizing effects by bristle thickness of toothbrushes than chlorhexidin. Reduction of bacterial colonization of toothbrushes might reduce the risk of maintaining bacterial infections of the upper respiratory tract. Accordingly, respective procedures are advisable, particularly as they are cheap and easy to perform.

Murine anti–third-party central-memory CD8+ T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells

AbstractTransplantation of T cell–depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donor-derived central memory CD8+ T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti–third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Blockade of VIP-Signaling Enhanced Anti-Leukemic Activity in Murine Allogeneic BMT without Significantly Increased GvHD.

Abstract 3001 Background and Objective:Vasoactive intestinal peptide (VIP) has potent immune-suppressive activity and can generate tolerogenic dendritic cells (DC) in vitro that block graft versus host disease (GvHD) in mouse model of BMT. We have previously published that absence of VIP signaling dramatically decreases PD-1 expression on activated CD8 T-cells and increases cellular antiviral immunity (JI 2011, 187:1057). To determine whether blockade of VIP-signaling enhances the graft-versus-leukemia (GvL) activity of donor T-cells in an allogeneic BMT model, we treated tumor bearing B6B′10BR allogeneic transplant recipients with a short course of daily s.c. injections of a small molecule VIP antagonist - VIPhyb or used VIP-knockout (VIP-KO) mice as BM donors. Methods:Recipient mice were inoculated with luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) by i.v. injection one day after lethal total body irradiation, then transplanted with the combination of 5 × 106 T cell-depleted BM (TCD-BM) plus splenocytes from either VIP-KO mice or wild-type (WT) littermates two days after irradiation. One group transplanted with WT BM and splenocytes received daily injections of 10 μg VIPhyb for one week; another group received saline injections. Survival, GvHD clinical sores (body weight, activity, posture, fur texture and skin), and bioluminescence imaging (BLI) were collected daily, twice a week, and weekly, respectively. Results:Transplantation of low dose (0.5 × 106) splenocytes from VIP-KO donors or low dose WT splenocytes in conjunction with VIPhyb-treatment dramatically improved tumor-free survival in the B6B′10BR allogeneic BMT model compared with PBS-treated recipients of WT grafts (Figure 1). The best overall survival (70%) and lowest number of mice with detectable tumor (10%) were seen in the VIPhyb-treated group. VIPhyb-treated mice did not have increased GvHD as assessed by clinical scoring. Recipients of VIP-KO grafts had 40% survival with no detectable tumors by BLI, and were without significant GvHD by clinical scoring. In contrast, the recipients transplanted with TCD-BM alone (without added splenocytes) and recipients that received 0.5 × 106 splenocytes and TCD-BM from WT donors and treated with PBS had increased tumor growth detected by BLI following BMT, and all of the mice died by 2 months post-BMT.Moreover, in non-tumor bearing mice transplanted with an intermediate dose (1 × 106) of splenocytes, survival was not different among recipients engrafted with VIP-KO BM and T-cells (84 ± 6 %), WT BM and T-cells treated with VIPhyb (89 ± 8 %) and WT BM and T-cells treated with PBS (94 ± 5 %). A similar enhancement of the GvL effect and a corresponding survival advantage for VIP-signaling blockade was seen in tumor-bearing transplant recipients of TCD-BM plus 1 × 106 splenocytes, with significantly better survival among recipients of VIP-KO donor cells (50%), recipients of WT cells treated with VIPhyb (60%) compared with recipients of WT cells treated with PBS (20%; p=0.04). Furthermore, in non-tumor bearing mice that received a higher dose (3 × 106) of splenocytes, recipients of VIP-KO BM and recipients of WT BM treated with VIPhyb had no significant increase in GvHD compared with recipients of WT BM treated with PBS (66 ± 9 %, 71 ± 8 % and 71 ± 8 % survival at 80 days, respectively). The mechanism by which administration of a VIP antagonist enhanced anti-tumor immunity includes the effect of blocking VIP-signaling induction of cAMP, leading to fewer Treg and fewer tolerogenic DC. Of note, blocking VIP-signaling led to significant decreases in expression of PD-1 and PD-L1 on CD8+ T-cells and DCs, respectively. Conclusion:Treatment with a small molecule antagonist of VIP-signaling, VIPhyb, dramatically increased anti-leukemic activity of donor T-cells without significantly increased GvHD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Enhances Anti-Tumor Activity After Transplant

Abstract 1897We have previously shown that haploidentical (HI) HSCT with low dose donor T-cell infusion provides a survival advantage in tumor bearing mice when compared to parent F1 or MHC-matched transplant models. We suggest that MHC difference in HI-HSCT generates early T-cell clonal activation against the unshared MHC haplotype, which eliminates residual tumor cells that express the unshared MHC haplotype. However, alteration in MHC antigen expression is a significant contributor to tumor escape from graft-versus-tumor (GVT) activity. Recent haploidentical transplant data revealed that uniparental disomy, the loss of the HLA haplotype, is a clinically relevant mechanism of tumor escape that leads to post-transplant leukemia relapse. Murine renal cell carcinoma, RENCA-TGL, cell line normally expresses only H2Kd as a MHC molecule. Therefore, in our haploidentical transplant model, T cell clonal activity is usually restricted against H2Kd molecule only. For circumventing the single haplotype expression of tumor model, we transfected this cell line with a H2Kb expressing vector, pAcGFP-NeoR-H2Kb, and generated stable clones with G418 selection. The clone that has more than 95% H2Kb expression used for in vivo experiments. Both tumor cell lines, i.e. parental and transfected clone, had similar in vivo tumor growth acceptance and growth rate. We then used two different haploidentical donors that were targeting different MHC haplotypes. Lethally irradiated B6D2F1 (H2Kb/d) mice were transplanted with T cell depleted bone marrow (TCD-BM) from either B6C3F1 (H-2Kb/k) (single haplo-1; SH1), or C3D2F1 (H2Kk/d) (single haplo 2; SH2) or both donor mice with low-dose (1×105) T-cells. In some experiments, animals were also injected either H2Kd or H2Kb/d expressing RENCA-TGL cells for the evaluation of GVT activity.Bone marrow (BM), spleens and thymi were harvested from recipients of single and double HI-HSCT at day 35 and showed similar cellularities. Interestingly, spleen and bone marrow had similar chimerism from both donors in DH-HSCT. There were no early transplant mortality, graft failure, weight loss and GVHD scoring difference among the double or single-haploidentical transplant recipients. In two other sets of experiments, we followed the tumor growth and the survival of tumor bearing mice after transplant. The recipients of DH-HSCT showed a better survival and GVT activity than the recipients of SH-HSCT in RENCA-TGL (H2Kb/d) bearing tumor model. These observations confirmed that MHC targeting plays a prominent role in tumor surveillance, and immune targeting the unshared MHC haplotype with haploidentical transplant induce remarkable survival advantage. Double HI-HSCT provides an unique anti-tumor activity that continues to exert GVT effect, even in case of MHC haplotype loss. Disclosures:No relevant conflicts of interest to declare.

Alternative Donor Hematopoietic Cell Transplantation for Patients with Fanconi Anemia.

AbstractAbstract 3081Hematopoietic cell transplantation (HCT) is the only proven curative therapy for the hematological abnormalities in patients with Fanconi anemia (FA). In the mid-1990s, survival after alternative donor (AD)-HCT was reported to be 18% with excessive rates of regimen-related toxicity, graft failure, graft-versus-host-disease (GVHD) and opportunistic infections (OI). Between 1990 and 2010, 127 FA patients underwent AD-HCT at the University of Minnesota using one of 5 consecutive, prospective phase I-II total body irradiation (TBI) containing clinical trials, representing the largest single center experience. All patients received cyclophosphamide (CY, 40 mg/kg) and single fraction TBI. Sequential changes were made to prevent GVHD, graft failure and OI and ultimately enhance survival; in 1994, all patients received T cell depleted (TCD) bone marrow (BM) or umbilical cord blood (UCB) to reduce GVHD; in 1999, fludarabine (FLU) was added to enhance engraftment; in 2003, thymic shielding (TS) was added to reduce OI risk; and in 2006, TBI dose reduction was evaluated to reduce toxicity. Over this time period, HLA-matched unrelated donor BM was the donor of choice with partially HLA matched BM and UCB only used as alternatives. Neutrophil recovery was twice as likely after FLU-containing regimens than non-FLU containing regimens and was not deleteriously affected by TS or reducing TBI dose to 300cGy. Grade II-IV acute GVHD was significantly reduced by the use of TCD (18%) compared to T replete BM or UCB (50% and 38%, respectively: p<0.01). Similarly, chronic GVHD was significantly lower with TCD BM (9%) vs T replete BM or UCB (25% and 15%, respectively; p=0.04). For the entire cohort of 127 patients, the probability of survival was 61% at 1 year and 54% at 5 years with a median follow-up of 9 years. Mortality was higher in recipients who were older (>10 years) or CMV seropositive, received any transfusions before HCT, or developed OI before HCT (Table). Mortality was lowest using CY/FLU/ATG, TBI 300 cGy with TS (Trial 5, relative risk [RR] 0.1; p<0.01). For 10 patients on Trial 5 who had no prior history of transfusions or OI, incidences of neutrophil and platelet are 100% and 100%, acute and chronic GVHD 17% and 0%, and probability of survival is 92%.Table:Factors associated with 3 year mortality: Multivariate analysisFactorsRelative Risk of Mortality (95% CI)P-valueTrial*2: CY/ATG/TBI 450-600, CSA/MP1.03: CY/FLU/ATG/TBI 450, CSA/MP0.3 (0.1–.7)<0.014: CY/FLU/ATG/TBI 450+TS, CSA/MP0.2 (0.1–0.7)0.015: CY/FLU/ATG/TBI 300+TS, CSA/MP or MMF0.1 (0.04–0.3)<0.01Donor TypeMatched URD Marrow1.0Mismatched URD Marrow0.3 (0.1–0.8)<0.01Mismatched RD Marrow0.3 (0.1–2.3)0.22Single or Double UCB2.0 (0.9–4.6)0.08Age at Transplant<10 years1.010–17 years2.4 (1.2–5.0)0.0118+ years3.1 (1.4–7.0)<0.01Prior OINone1.0Yes3.2 (1.5–6.9)<0.01Prior TransfusionsNoneYes2.4 (1.0–5.8)0.05CMV SerostatusPatient negative/donor negative1.0Patient negative/donor positive1.5 (0.7–3.4)0.34Patient positive2.2 (1.1–4.3)0.03*Trial 1 (CY/TBI 450, MTX/MP or CSA) was excluded as there were only 8 patients in this group and a number of demographic and disease factors were unknown.Substantial progress has been made in the successful application of AD-HCT for FA. These data support the urgency of transplant prior to the onset of severe pancytopenia when risk of OI and transfusions are more likely, particularly when a suitable BM donor is available. While other regimens are being explored, CY/FLU/ATG/TBI300 with TS should be considered a new standard of care for FA patients undergoing AD-HCT. Disclosures:No relevant conflicts of interest to declare.

Donor Treatment with α1 Anti-Trypsin (AAT) Mitigates Gvhd and Increases Survival While Sparing GVL Effects

Abstract 1889 Background:Graft-versus-host disease (GVHD) is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). While cytolytic effector function of donor-derived CD8+ cytotoxic T lymphocytes (CTLs) is critical for the elimination of the underlying malignant disease, the goal is to achieve this graft-versus-leukemia (GVL) effect without inducing GVHD. We and others showed in murine models that administration of AAT in the peri-transplant period decreased GVHD incidence and mortality. Concurrently, levels of pro-inflammatory cytokines were suppressed compared to albumin-treated recipients. Since both GVHD and GVL effects are initiated by donor cells, we asked whether donor treatment with AAT would modify GVHD or GVL effects in recipients. Of concern was the observation that AAT enhanced expression of heme oxygenase-1 (HMOX-1) which, via generation of carbon monoxide, enables the transcription factor Nrf2 to bind to the promoters of IL10 and IL1Ra, which might interfere with GVL activity. Methods:We determined the effect of donor AAT treatment on GVHD and GVL in two murine transplant models: C3H/SW(H-2Kbc) into C57Bl/6(H-2Kb) (minor mismatch), and C57Bl/6(H-2Kb) into BALB/C (H-2Kd) (major mismatch). Four regimens of donor and recipient treatment were studied: 1) Donor albumin /recipient albumin; 2) donor albumin/recipient AAT; 3) donor AAT/recipient albumin; 4) donor AAT /recipient AAT. AAT and albumin were given to donors at 3 μg/ dose every 72 hours for two weeks before cell harvesting. Recipients received 3 μ/dose every 72 hours following total body irradiation with 800 cGy. On day 4, recipients were injected with 5 × 106 T cell-depleted bone marrow (TCD-BM) plus 5 × 105 CD4+/CD8+ splenic T cells to induce acute GVHD. To test our hypothesis that donor AAT treatment would interfere with the GVL effect, BALB/C (H-2Kd) recipients were injected i.v. with 20 × 103 A-20 lymphoma cells containing a luciferase reporter construct (A20-luc/yfp; H-2Kd), following TBI. The infusion of TCD-BM and T cells from C57Bl/6(H-2Kb) donors was delayed until day 4 to allow the tumor to home and get established. Results:RT-PCR analysis of donor BM and T cells prior to transplantation showed up-regulation of HMOX-1 and Nrf2 (10 and 15 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively, compared to albumin treated donors). In contrast, TNFa, was down-regulated (7 log2 decrease in comparison to albumin-treated donors) in both unsorted bone marrow and in CD8+/CD4+spleen cells from AAT pre-treated donors. FACS analysis revealed a 4-fold increase in frequency of CD56+NK cells, and a 5-fold increase in CD4+/CD24+FoxP3+ Treg cells, associated with a 3 fold increase in CD8+/CD205+ APCs/ DC in comparison to albumin treated donors. Mice transplanted with cells from AAT-treated donors showed lower clinical GVHD scores and decreased mortality (Figure 1) in comparison to mice transplanted from albumin-treated donors. BALB/C (H-2Kd) animals transplanted from AAT-treated donors showed significantly lower A-20 tumor signal (luciferase) intensity (over the first 3 weeks post-HCT) than did mice transplanted from albumin-treated donors (n=6, mean 4.8 × 106 (AAT) versus,7 × 106 (albumin) photons/sec/mouse, P =.03). Summary and conclusions:These data suggest that pre-transplant in vivo AAT exposure of donor cells results in protection against acute GVHD. Tumor growth was not increased in mice transplanted from AAT treated donors and, in fact, appeared to be suppressed, presumably related to a significant increase in NK cells. Further studies on donor pre-treatment or direct administration of AAT to the transplant recipients are warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clofarabine Cytoreduction Followed by a Second Transplant Using Haploidentical Donors for Relapsed Acute Leukemia After First Allogeneic Hematopoietic Stem Cell Transplantation: Early Outcome, Infection and Toxicity.

AbstractAbstract 3071▪▪This icon denotes a clinically relevant abstract Background:Prognosis of relapse after first allogeneic hematopoietic stem cell transplantation (allo-HSCT) is poor, and no standard treatment is available. Second HSCT (HSCT2) is a valuable treatment option for selected patients. Change of donor for HSCT2 is a frequently used strategy to enhance the graft-versus leukemia-activity, although its role for improving the outcome is still unclear. However, identification and activation of a new donor for HSCT2 is time consuming and might delay or even prevent a second transplant. In contrast, haploidentical family donors are rapidly available in the vast majority of patients and might therefore be an alternative for HSCT2, once change to a new donor is planned.Based upon these considerations, HSCT2 from haploidentical family donors was used as preferred treatment for acute leukemia relapse after allo-HSCT in our transplant center since August 2009. According to the concept of sequential therapy (as introduced by the FLAMSA-RIC regimen), patients initially received cytoreductive chemotherapy, followed, after three days of rest, by reduced intensity conditioning (RIC). Based on the Baltimore protocol for haploidentical HSCT using RIC and post-transplant cyclophosphamide for depletion of allo-reactive T-cells, individual and disease-specific modification of this regimen were used for preparation before HSCT2. Patients and Methods:Fourteen adult patients (7 male, median age: 37 years) suffering from AML (n=11) or ALL (n=3) with an ECOG score of <=1 and with a partially HLA-matched (haploidentical) family donor available, were eligible. They had relapsed after first (n=12) or second (n=2) HLA matched related (n= 5), matched unrelated (n=8) and partially matched related (n=1) allo-HSCT. Median duration of remission prior to haploidentical HSCT2 was 400 days (range 45–1701). Median interval between relapse and HSCT2 was 78 days (range 27–205). Initially all patients received cytoreductive chemotherapy with clofarabine (30mg/kg IV on day -15 to -11), and conditioning treatment consisted of fludarabine (30 mg/m2 IV on day -7 to -3) and cyclophosphamide (14,5 mg/kg, IV on day -7 to -6) plus either melphalan (110 mg/m2 IV on day -2, n=9) or treosulfan (3×10 g/m2 IV on day -4 to -2, n=3) or TBI (200 or 400 cGy on day -1, n=2) followed by a non T-cell depleted bone marrow infusion on day 0. Post-grafting immuno-suppression consisted of cyclophosphamide (50mg/kg IV on day +3 and +4) plus tacrolimus (days 5–180) and mycophenolate mofetil (days 5–35). Results:Neutrophil and platelet engraftment was achieved in 12 (85,7%) and 10 (71,4%) patients, respectively. Median time to neutrophil engraftment was 32 days (range 24–90). No primary graft rejection was observed. Complete remission could be induced by day +30 in 11 patients (78,6%), whereas 1 patient died early and 2 patients had refractory disease. Full donor chimerism by day +30 was achieved in 10 patients (71,4%).After a median follow-up of 6 months (range 4 days–33 months) cumulative incidence of non-relapse mortality at day + 30, + 100 and +360 was 7%, 24% and 24 %, respectively. Rate of grade II-IV acute GvHD was 24,7%. Mild or moderate chronic GvHD was seen in 2 patients (14,3%). 8 patients died from relapse (n=4), infection (n=3) or infection and toxicity (n=1). Estimated overall survival and progression-free survival at one year from HSCT2 was 53 % (+/− 14 %) and 36 % (+/− 17 %), respectively.Infection was frequent detected in 13 (92,9%) patients with at least one episode of neutropenic fever in 8 patients, probably or proven invasive aspergillosis in 5 patients and a moderate rate of virus-reactivation/-infection (CMV 21,4%, HSV 14,2%, EBV 28,6%, HHV-6 28,6%, Polyomavirus 28,6%, Adenovirus 21,4%,). No patient developed a post-transplant lympho-proliferative disorder.Regimen related grade III-IV toxicity was observed in 9 (64,3%) patients with transient elevation of liver enzymes in 7 (50%), mucositis in 5 (35,7%), headache in 5 (35,7%), hand-foot syndrome in 3 (21,4%), hyper-bilirubinemia in 3 (21,4%) and severe deterioration of kidney function in 2 (14,3%) patients, respectively, as the most common observed toxicities. Conclusion:Haploidentical HSCT2 following initial cytoreduction with clofarabine, RIC and high-dose cyclophosphamide post-transplant is feasible in patients with acute leukemia relapsing after first allo-HSCT. Early results are promising, but long-term results are still pending. Disclosures:Off Label Use: Clofarabine is a novel purine nucleoside with immunosuppressive and anti-leukemic activity in hematologic malignancies, off-label use in adults.

FTY720 Markedly Increases Alloengraftment but Does Not Eliminate Host Anti-Donor T Cells that Cause Graft Rejection on Its Withdrawal

The immunomodulator FTY720 (FTY) is beneficial in models of graft-versus-host disease, solid organ transplantation, and autoimmunity and has been approved for use in patients with multiple sclerosis. FTY modifies the homing and migration of many cell types. We report that FTY has profound positive and negative effects on allogeneic bone marrow (BM) engraftment in sublethally irradiated recipients. FTY increased donor hematopoietic progenitors in the BM, resulting in high donor engraftment in the B cell, myeloid cell, and natural killer cell, but not T cell, lineages. Donor T cell progenitors within the thymus of FTY-treated recipients were dramatically reduced, resulting in a lack of donor T cell reconstitution. In addition to preventing the ingress of donor (and host) T cell progenitors, FTY prevented the egress of fully functional host CD4+CD8- and CD4-CD8+ thymocytes that on cessation of FTY administration were able to exit from the thymus and contribute to a rapid and complete rejection of a well-established donor BM graft. When used in combination with anti-CD40L mAbs to block the CD40L:CD40 costimulatory pathway, FTY markedly enhanced anti-CD40L mAb-mediated alloengraftment promotion. In contrast to FTY alone, the combination of anti-CD40L mAb and FTY resulted in a surprisingly stable, multilineage, long-term donor chimerism. These data illustrate FTY's profound migration modulating effects and suggest a use in combinatorial therapy in achieving stable alloengraftment under nonmyeloablative conditions.

Donor but Not the Host Derived Interleukin-10 (IL-10) Contributes to Regulation of Graft-Versus-Host Disease (GVHD)

Abstract 2975IL-10 is a key immune-regulatory cytokine and its gene polymorphisms correlate with severity of clinical graft-versus-host (GVHD). IL-10 is made by a variety of donor and host cells, but the functional relevance of its source in acute GVHD is not well understood. We utilized well characterized MHC matched, minor mismatched C3H.SW →B6 model of acute GVHD to first examine the relevance host hematopoietic derived IL-10 on acute GVHD induced by alloreactive donor T effector cells in the absence of donor regulatory T (Tregs) cells. We generated [WTB6→B6-Ly5.2] and [IL-10−/−B6→B6-Ly5.2] chimeras such that only the host hematopoietic derived cells were incapable of producing IL-10 and utilized them as recipients. The chimeras were lethally irradiated and transplanted with 5×106 T cell-depleted (TCD) BM and 2×105 CD8+ T cells from either the allogeneic C3H.SWor the syngeneic B6 donors. All of the allogeneic animals demonstrated similar clinical severity and mortality from GVHD (65% vs 72%, P=NS). Because IL-10 can also be derived from non-hematopoietic cells, we next determined whether the absence of IL-10 secretion by both hematopoietic and non-hematopoietic cells would modulate GVHD severity. To this end, the WT B6 and the IL-10−/− B6 animals were irradiated with 10Gy and transplanted 5×106 T cell-depleted bone marrow (TCDBM) and 2×105 CD8+ T cells from either allogeneic C3H.SW or the syngeneic B6 donors. All of the allogeneic animals demonstrated similar severity and mortality from GVHD regardless of whether all of the host cells can make IL-10 (P=0.1, NS).We next examined relevance of IL-10 deficiency in donor cells. 1×106 CD25+depleted donor T effectors from either WT or IL-10−/− B6 mice were infused along with 5×106 TCDBM from WT B6 into lethally irradiated allogeneic C3H.SW or syngeneic B6 animals. The allogeneic animals that received either WT or IL-10−/− B6 effector cells demonstrated similar GVHD severity and mortality (P=0.6). Further, when T effectors from WT B6 mice were infused along with TCDBM from either WT or IL-10−/− B6 donors, the allogeneic C3H.SW animals once again demonstrated similar GVHD mortality (70% vs 80%, P=NS). Thus deficiency of IL-10 in either host tissues or in the donor grafts did not alter CD25+ depleted donor T effector cell mediated severity of GVHD.We next examined the relevance of the source of IL-10 in donor natural (Treg) mediated regulation of GVHD. Donor CD4+CD25+ Tregs from C3H.SW donors induced equivalent protection from GVHD mortality in both WT and IL-10−/− hosts (90% vs. 100%, P=NS). To examine the relevance of IL-10 derived from Tregs, we transplanted 0.25×106 CD4+CD25+ Tregs from either B6 or IL-10−/− donors along with 1×106 CD25 depleted T effectors (1 :4 ratio) into C3H.SW animals. 100% of the allogeneic animals that received WT donor Tregs survived while only 60% of those that receive IL-10−/− donor Tregs survived (P=0.001). However, when the Il-10−/− donor Treg numbers were increased to 1×106 (1 :1 ratio), all of the allogeneic animals survived (P=0.008) demonstrating that donor Treg derived IL-10 is not obligatory but is necessary for optimal reduction of GVHD.To determine the role of donor BM derived IL-10 in Treg mediated protection from GVHD, Tregs and T effector cells from WT B6 donors (1 :4 ratio) were transplanted along with 5×106 TCD BM from either WT or IL-10−/− B6 donors into lethally irradiated allogeneic C3H.SW animals. The allogeneic recipients of WT BM were protected from GVHD mortality, donor Tregs failed to protect the allogeneic animals that received IL10−/− donor BM (85% vs. 20%, P<0.04) in whom the rate of mortality was similar to the animals that did not receive any donor Tregs (P=0.96). Mechanistic studies demonstrated that donor Tregs continued to express similar levels of foxp3 and expansion early (day 7) after BMT. However the their expansion was significantly reduced at later time-points (day 14 and 21) when the hosts received BM from IL-10−/− donors. Together these data suggest that IL-10 from either host or donors does not contribute to the induction of GVHD severity by the T effector cells. However, only donor BM and donor Treg derived IL-10 is important for donor Treg mediated suppression of GVHD. Disclosures:No relevant conflicts of interest to declare.

IL-15 Administration Increases Graft Versus Leukemia Activity in Recipients of Haploidentical Hematopoietic Stem Cell Transplantation,

Abstract 4027The success of haploidentical (HI) hematopoietic stem cell transplantation (HSCT), suggests that graft-versus-leukemia (GVL) effect might have a substantial role in this transplant modality. Rigorous T-cell depletion (TCD) of the graft decreases the occurrence of graft-versus-host disease (GVHD) in HI-HSCT, however this results in immunodeficiency and high disease relapse rate, especially in patients with resistant or residual leukemia. Therefore, enhancing GVL activity of HSCT without increasing GVHD is crucial for improving the outcome of haploidentical transplant. Post-transplant IL-15 administration is shown to enhance immune reconstitution, particularly donor-derived NK and CD8+ T cell populations in murine models. We evaluated the efficacy of IL-15 for enhancing GVL effect in recipients of HI-HSCT.For developing clinically relevant haploidentical transplant models, different hybrid mice with B6 background that share the same haplotype (H2Kb) are used for our murine haploindentical transplant experiments. Lethally irradiated B6D2F1/J (H2Kb/d) mice are transplanted with B6CBAF1/J (H2Kb/k) TCD bone marrow (BM) and T cells at varying doses. Some animals were also given P815 tumor cells on the day of transplant. Administration of IL-15 significantly increased the numbers of CD8+ T and NK cells in the spleen and BM in the T cell depleted model at post-transplant day 28. Infusion of very low dose haploidentical T cells (1×104) with TCD-BM resulted in a conflicting effect on immune reconstitution, i.e. increased T cell numbers, and decreased NK cell population. Post-transplant IL-15 administration also changed this immune reconstitution pattern and significantly increased both T and NK cell numbers in recipients of HI-HSCT. In P815 challenged mice that were transplanted with very low dose T cell added TCD-BM, IL-15 administration significantly increased anti-tumor activity of the graft and improved survival (Figure 1) without increasing GVHD. This effect was observed when IL-15 administration was given at a later time point rather than immediately following transplantation, possibly allowing for more donor cell engraftment and T cell proliferation to take place. IL-15 administration without T cell infusion did not result in any survival improvement. We conclude that in our experimental HI transplant models, IL-15 administration augments anti-tumor effect of the HI-HSCT without increasing GVHD risk, and this effect requires presence of donor derived T cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Alloreactivity Directed Against the Widely Distributed HY Antigen Impairs Antitumor Immunity and Results in T-Cell Dysfunction

Abstract 2964 INTRODUCTION:The curative potential of allogeneic transplant for high-risk malignancy is based on the observation that alloreactivity can result in a clinically significant graft-versus-tumor (GVT) effect. However, we have observed that alloreactivity directed against non-tumor restricted miHA's reduces quantitative responses to vaccines targeting tumor-specific antigens. The relative impact of the GVHD-mediating antigen on the potency of the GVT response when the antigen is shared has not been well studied. METHODS:A murine allotransplant system in which the clinically relevant GVHD antigen HY drives both graft-versus-host disease and the antitumor response was utilized. Following lethal irradiation, combinations of B6 male (HY-expressing) and female (HY-naïve) donors and recipients were used in T-cell depleted bone marrow transplants to control for HY expression in hematopoetic and non-hematopoetic compartments. Delayed donor lymphocyte infusion (DLI) with female HY-specific transgenic T-cells was then performed which allowed tracking of antigen-specific cells. Mice were subsequently challenged with an immunogenic HY-expressing tumor (MB49). In tumor protection studies, transplant recipients received a male dendritic cell vaccine at the time of DLI. Recipients were monitored for clinical GVHD scoring, weight loss, tumor-free and overall survival. Surface phenotyping of HY-specific CD8 T cells from recipient bone marrow, tumor-draining lymph node (LN) and spleen was performed serially by flow cytometry using congenic markers. Statistical analyses were performed using paired Student t-tests and Kaplan-Meier survival estimates. RESULTS:Transplantation of female marrow and HY-specific T cells into male recipients produces a mild HY-targeted GVHD, indicated by weight loss and skin GVHD scores. Female recipients of female marrow and HY-specific DLI had 100% survival following HY-expressing tumor challenge. In contrast, male recipients had only 20 +/− 4.7% tumor-free survival (p<0.0001), despite receiving HY-reactive female marrow and HY-specific DLI. Administration of an HY-expressing male dendritic cell vaccine did not improve either tumor growth velocity or tumor-free survival in male recipients. Despite a poor antitumor response in males, expression of HY on nonhematopoetic tissues produced a significant expansion of HY specific T-cells following DLI, regardless of tumor-bearing status (30.5 −77.4% total CD8 from spleen, draining LN and marrow, vs 0.01–1% from female recipient controls, p<0.0001). This suggested that impaired tumor control was due to dysfunction, rather than deletion, of HY-specific T cells. Indeed, nearly 100% of HY-specific CD8 isolated from the spleen, tumor-draining lymph node, and bone marrow of male recipients expressed high levels of PD-1, a phenomenon observed at all time points in tumor-bearing and non tumor-bearing male recipients with HY-directed GVHD. Non-HY specific CD8 cells did not express PD-1 (p<0.0001). Further, HY-specific CD8 from spleen and tumor-draining LN of male recipients display a significantly increased percentage of CD44+CD62L- effector memory (72.4 +/− 17.2%) vs. CD44+CD62L+ central memory (15.9 +/− 9.7%, p= 0.006) cells compared to non-HY specific CD8 cells (26.5% +/− 2.8 % vs. 28.2 +/− 12.7%, p= 0.52) from male and female recipient controls. CONCLUSIONS:In an experimental system where HY is expressed on both recipient nonhematopoetic tissue and tumor, HY-directed alloreactivity impairs the antitumor response despite antigen-specific DLI and effective vaccination. Characterization of alloreactive CD8 T cells in this setting reveals a persistence of effector memory and high levels of PD-1 expression, which suggest T-cell dysfunction as a possible mechanism. Further studies of T-cell dysfunction in this model may identify targets for therapeutic blockade following adoptive immunotherapy with particular relevance to those clinical situations where GVHD does not enhance GVT. Disclosures:No relevant conflicts of interest to declare.

Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Abstract 144In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT.Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus.To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients.Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures:No relevant conflicts of interest to declare.