Alloreactive Cells Research Articles

Placenta-derived decidual stromal cells – a novel therapy for graft-versus-host disease, haemorrhages and toxicity after allogeneic haematopoietic stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (ASCT) is a well-established therapy for leukaemia and other disorders of the immunohaematopoietic system. However, transplanted cells can attack the patient ’ s tissue, so-called graft-versus-host disease (GVHD). Severe GVHD can lead to death. Other complications after ASCT include toxicity,haemorrhages and infections. Stromal cells, such as mesenchymal stem cells (MSCs) or placenta-derived decidual stromal cells (DSCs), have an immunosuppressive and anti-inflammatory effect. This effect is seen despite human leucocyte antigen (HLA) incompatibility between the stromal cells and stimulatory or alloreactive cells in vitro , which enables the use of third-party stromal cells in the clinic. I was the first to use MSCs for life-threatening acute GVHD with miraculous response in some, but not all, patients.

Graft-versus-host disease, a major complication after stem cell transplantation – home care for prevention and stromal cells for therapy

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic haematopoietic stem cell transplantation (HSCT). Although severe acute GVHD is associated with a high mortality, chronic GVHD is associated with a strong anticancer effect. Patients suffering from leukaemia who undergo HSCT have a reduced risk of recurrent disease if they develop chronic GVHD. During the neutropenic phase after HSCT, patients are isolated in the hospital, sometimes in laminar airflow rooms. We challenged this by allowing patients to be treated at home. A nurse from the unit visited and checked the patient daily. Compared with hospital controls, home-care patients had a decreased risk of acute GVHD, which was associated with number of days treated at home during neutropenia. Home care did not affect chronic GVHD or relapse and, therefore, home-care patients had a trend for better survival than those treated in the hospital. Stromal cells such as mesenchymal stem cells (MSCs) or placenta-derived decidual stromal cells suppress alloantigen-induced T-cells in vitro. This effect was seen despite HLA incompatibility between MSCs and stimulatory or allo-reactive cells in vitro, enabling the use of third-party stromal cells in the clinic. Because stromal cells home to target organs of tissue toxicity, and have low immunogenicity, they may be useful in regenerative medicine such as acute and chronic GVHD, haemorrhages and tissue toxicity. I was the first to use MSCs for life-threatening acute GVHD with miraculous response in some, although not all, patients. Clinically, MSCs were found to completely reverse severe acute GVHD in approximately 50% of patients. Among patients with chronic GVHD, the complete response rate was approximately 25%. Stromal cells interfere with coagulation and reversed haemorrhagic cystitis and may stop major haemorrhages in patients who have undergone HSCT. In addition to GVHD and haemorrhages, stromal cells may be used for treatment of graft failure and allograft rejection in organ transplant patients, for tissue toxicity in cancer patients and to treat autoimmune disorders. To conclude, GVHD is a limiting severe side-effect of HSCT which may be decreased by home care. Stromal cells have potential in regenerative medicine and have healed severe GVHD, although improvements are warranted.

Natural killer cells and regulatory T cells: how to manipulate a graft for optimal GVL

Two of the major complications that limit the efficacy of allogeneic hematopoietic cell transplantation (allo-HCT) are disease relapse and GVHD. Due to their rapid recovery early after allo-HCT and their ability to kill malignant targets without prior exposure, natural killer (NK) cells have been considered one of the main effector cells that mediate early GVL reactions. Conversely, regulatory T ells (Tregs) have proven to be critical in facilitating self-tolerance. Both murine and human studies have demonstrated a significant role for Tregs in the modulation of GVHD after allo-HCT. This article reviews the mechanisms of how these 2 cell types carry out these functions, focusing on the post-allo-HCT period. Surprisingly, relatively few studies have addressed how Tregs and NK cells interact with one another and whether these interactions are antagonistic. Although preclinical studies suggest active cross-talk between NK cells and Tregs, early clinical studies have not shown a detrimental impact of Treg therapy on relapse. Despite this, interruption of tolerogenic signals may enhance the efficacy of NK effector functions. Methods to transiently impair Treg functions and augment NK cell alloreactivity will be discussed.

The Cholinergic Anti-Inflammatory Pathway Delays TLR-Induced Skin Allograft Rejection in Mice: Cholinergic Pathway Modulates Alloreactivity

Activation of innate immunity through Toll-like receptors (TLR) can abrogate transplantation tolerance by revealing hidden T cell alloreactivity. Separately, the cholinergic anti-inflammatory pathway has the capacity to dampen macrophage activation and cytokine release during endotoxemia and ischemia reperfusion injury. However, the relevance of the α7 nicotinic acetylcholine receptor (α7nAChR)-dependent anti-inflammatory pathway in the process of allograft rejection or maintenance of tolerance remains unknown. The aim of our study is to investigate whether the cholinergic pathway could impact T cell alloreactivity and transplant outcome in mice. For this purpose, we performed minor-mismatched skin allografts using donor/recipient combinations genetically deficient for the α7nAChR. Minor-mismatched skin grafts were not rejected unless the mice were housed in an environment with endogenous pathogen exposure or the graft was treated with direct application of imiquimod (a TLR7 ligand). The α7nAChR-deficient recipient mice showed accelerated rejection compared to wild type recipient mice under these conditions of TLR activation. The accelerated rejection was associated with enhanced IL-17 and IFN-γ production by alloreactive T cells. An α7nAChR-deficiency in the donor tissue facilitated allograft rejection but not in recipient mice. In addition, adoptive T cell transfer experiments in skin-grafted lymphopenic animals revealed a direct regulatory role for the α7nAChR on T cells. Taken together, our data demonstrate that the cholinergic pathway regulates alloreactivity and transplantation tolerance at multiple levels. One implication suggested by our work is that, in an organ transplant setting, deliberate α7nAChR stimulation of brain dead donors might be a valuable approach for preventing donor tissue inflammation prior to transplant.

Selective organ specific inflammation in offspring harbouring microchimerism from strongly alloreactive mothers

The origins of autoimmunity are not yet understood despite significant advances in immunology. The trafficking of maternal cells to the offspring represents the very first immunological event in foetal life and is reinforced during lactation. The persistence of maternal cells in offspring's tissues and circulation has been associated with several autoimmune disorders. However a direct causal effect has never been demonstrated. Maternal T cells specifically targeting foetal insulin producing cells have been shown to generate islet inflammation without directly participating in this process. Our objective was to evaluate if alloreactive maternal cells could directly trigger a graft-versus host like reaction or indirectly influence the development of the offspring's regulatory T cells favouring autoimmunity. We adopted a breeding strategy comparing genetically identical offspring from either strongly alloreactive transgenic mothers compared to immunodeficient mothers. We detected maternal alloreactive T cells in the offspring and early signs of inflammation in small intestine of 6 weeks old offspring. Interestingly, CD4+ Foxp3+ regulatory T cell frequency was diminished in mesenteric lymph nodes from eight months old offspring born of alloreactive mothers compared to offspring of immunodeficient mothers. Our study favours a hypothesis where highly alloreactive maternal cell microchimerism indirectly predisposes offspring to autoimmunity.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant.Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures:No relevant conflicts of interest to declare.

Reconstitution Of NK Cells After Reduced Intensity Conditioned Unrelated Cord Blood Transplantation In Patients With Acute Myeloid Leukemia: Analysis Of a Prospective Phase II Multicentric Trial On Behalf Of Societe Française De Greffe De Moelle Osseuse Et Therapie Cellulaire (SFGM-TC) and Eurocord

Background Unrelated cord blood transplantation (UCBT) after reduced intensity conditioning regimen (RIC) has extended the use of cord blood in elderly patients and those with co-morbidities without an HLA identical donor, although relapse post transplant remains a concern in high risk AML patients. HLA incompatibilities between donor and recipient might enhance Natural Killer (NK) cell alloreactivity after allogeneic hematopoietic stem cell transplantation (HSCT). We previously observed that the quality of NK cell reconstitution was impaired after haploidentical HSCT, impacting on graft versus leukemia (GvL) effect, but was preserved after UCBT in a small cohort of patients. Methods To evaluate RIC-UCBT in patients with acute myeloid leukemia (AML), a prospective phase II multicentric trial was conducted in France, whose primary objective was to show a reduction in non-relapse mortality (NRM) from 40% (based on registry data) to 20%. Seventy-nine patients were enrolled for a de novo or secondary AML in complete remission (CR). The conditioning regimen consisted of cyclophosphamide (50mg/kg) + fludarabine (200mg/m2) + total body irradiation (2Gy), CsA +MMF as GVHD prophylaxis and GCSF from day +1. Patients were enrolled in 23 centers from October 2007 to September 2009. Engraftment rate was 87 % at day+60. At 2 years, overall survival, incidence of relapse and LFS were respectively 44%, 46% and 35%. Peripheral blood samples were collected following UCBT in order to realize an extensive phenotypic and functional study of NK cells. Studies were started at 1 month (M1) post UCBT with available samples for 62 out of the 69 included patients, and were compared to 20 healthy donors and 15 cord blood (CB). Results Total CD3+ T-cells were 117 /mm3 at M1 (range 0-934), and 465 /mm3 at M3 (range 0-2917). CD19+ B-cells were 36/mm3, (range 0-524) and 342/mm3 (range 0-2990) at M1 and M3 respectively. NK cell recovery was prompt, representing 47% of the total lymphocyte population at M1 (186 CD3-CD56+ NK cells/mm3), 30% at M3 (239/mm3; range 2-767) and decreasing to normal rate at M6 (20% of lymphocytes). At M1 post-UCBT, NK cells exhibited high rate of CD56bright, NKG2A, and KIR2DL4 associated with a decreased expression of CD8 and CD161, compared to CB and healthy donors. These immature characteristics were transient and return to normal value from M3 or M6 post-UCBT. Interestingly, we also observed a significant increased expression of the activation markers CD69, and HLA-DR during the whole period of the study, compared to CB and healthy donors, which probably reflects a persistent proliferation state of the NK cells. On the other hand, NK cells post-UCBT were indistinguishable from CB and healthy donors control samples for other receptor tested such as NKp30, NKp46, NKp80, and NKG2D. Notably, Expression of KIR2DL1 was decreased at M1 and M3 but reached similar values to controls at M6, whereas, KIR3DL1 was increased during the whole study. To determine the significance of these phenotypic features, we assessed polyfunctional ability of NK cells following UCBT by a combined analysis of the degranulation (CD107a), and the production of IFN-γ and TNF-α. This study reveals that NK at M1 post-graft exhibited a transient higher ability to produce IFN-γ than healthy donors (p<0.0001), which reaches normal values by 6 months after UCBT, in correlation with the evolution of the immunoregulatory NKG2A+/CD56brightNK cells subset post transplant. Production of TNF-α was reduced in CB and at M1 as compared to healthy donors (p<0.0001) but quickly restored starting from M3. Degranulation’s ability was slightly impaired at M1 and M3, as compared to CB and healthy donors (p=0.002), but restored at M6. Conclusion This study shows that after RIC-UCBT, NK cells display some phenotypic features of activation associated with a prompt and complete restoration of their ability for polyfunctional activities. Further analyzes are needed to assess the impact of such observation on NK cell mediated GvL effect in this prospective trial of RIC-UCBT for AML patients in CR. Disclosures: No relevant conflicts of interest to declare.

Missing HLA C Group 1 Ligand In Patients With AML and MDS Is Associated With Reduced Risk Of Relapse After Allogeneic Stem Cell Transplantation With Fludarabine and Treosulfan Reduced Toxicity Conditioning

Allogeneic stem cell transplantation (SCT) is a curative therapy for patients with AML and MDS. Reduced toxicity conditioning with fludarabine and treosulfan (FT) has been shown to be a dose intensive regimen with enhanced anti-leukemia effect and acceptable toxicity. However, relapse after SCT remains the main obstacle to cure. Natural killer (NK) cell alloreactivity has a documented role in reducing relapse risk after haplotype mismatched SCT in AML. The role of NK alloreactivity in HLA matched SCT is more controversial. The missing ligand theory suggests that missing ligands in the recipient for donor inhibitor Killer immunoglobulin-like receptors (KIR) may drive NK alloreactivity after SCT in the absence of HLA mismatch. In this analysis we defined the prognostic factors for relapse and overall survival (OS) in patients with AML and MDS given FT conditioning. In particular we tested the role of recipient HLA ligands for NK KIRs in controlling relapse. The study included 203 patients with AML (n=129, 29 of them secondary to MDS or prior chemotherapy) and MDS (n=74) given SCT in 2 institutions. The median age was 58 years (range, 21–76), 126 male, 77 female. The donor was an HLA-matched sibling (n=97) or matched unrelated (n=106). Disease status was CR1 (n=65), CR2/later CR (n=24), no CR (n=44) or previously untreated MDS (n=70). Eighty patients (39%) had poor-risk cytogenetic or molecular markers. FT included treosulfan 12 gr/m2 x3 days in one institution (FT12, n=123) or 14 gr/m2 x3 in the second (FT14, n=80). 135 patients with an unrelated donor or no prior therapy were also given ATG. High-resolution HLA typing revealed that 67% expressed at least one Bw4 antigen, 81% expressed group 1 C alleles and 66% group 2 alleles. With a median follow-up of 48 months (range, 6-108 months) 86 patients are alive, 51 died of non-relapse causes (NRM) and 66 died of relapse. Seven additional patients relapsed but are currently alive. The 5-year OS and leukemia-free survival (LFS) rates were 39% (95%CI, 32-47)and 36% (95%CI, 28-43), respectively. The 5-year cumulative incidence of relapse and NRM was 38% (95%CI, 31-45) and 27% (95%CI, 31-45), respectively. The most significant predictor of relapse was the status of disease at SCT. 5-year relapse rates were 33% (95%CI, 26-42) and 55% (95%CI, 42-71) for patients in CR/untreated or refractory disease, respectively (p=0.001). Patients expressing 1-2 HLA C group 1alleles had a relapse rate of 45% (95%CI, 37-56) compared to 26% (95%CI, 13-49)in patients with missing HLA C group 1 ligand (p=0.03). Missing HLA C group 2 and/or Bw4 ligands had no effect on relapse. Multivariate analysis identified no CR at SCT (HR 3.6, p=0.001), missing HLA C group 1 ligand (HR 2.6, p=0.03), sibling donor (HR 1.8, p=0.04), poor cytogenetics (HR 1.7, p=0.05) and female donor to male recipient (HR 0.5, p=0.06) as independent factors predicting relapse. The reduced relapse rate associated with missing HLA C group 1 ligand was more pronounced in patients with MDS/secondary AML, 39% Vs 7%, respectively (p=0.02) and in patients not in CR or in untreated disease at SCT, 46% Vs. 8%, respectively (p=0.02). Missing HLA ligands were not associated with GVHD or NRM. The predicting factors for NRM were SCT beyond CR1 (HR 8.6, p=0.0002), Sibling donor (HR 0.6, p=0.08) and SCT comorbidity score > 2 (HR 1.8, p=0.08). The reduced relapse rate combined with no effect on NRM of missing HLA C group1 ligand translated into improved LFS, been 46% (95%CI, 27-65) compared to 30% (95%CI, 21-40) in patients expressing the ligand (p=0.07). Multivariate analysis identified SCT not in CR (HR 2.8, p=0.0007), SCT comorbidity score > 2 (HR 1.5, p=0.06) and missing HLA C group 1 ligand (HR 1.9, p=0.02) as independent predicting factor for LFS. In conclusion, missing HLA C group 1 ligand in SCT recipients with AML and MDS may be associated with reduced relapse risk, similar NRM and improved LFS. This effect is more pronounced in patients with MDS and secondary AML and in patients with advanced disease. The speculated mechanism is enhanced NK alloreactivity in this setting according to the missing ligand theory. These observations merit further study in larger cohorts and in patients given other conditioning regimens. They may also impact planning of immune therapies after SCT. Disclosures: No relevant conflicts of interest to declare.

The Hypoxia Inducible Factor-2/Arginase-1 Axis Plays An Adaptive Role In Gvhd

The intestinal epithelium is a primary target of graft-versus-host disease (GVHD). The hypoxia-signaling pathway has been implicated as an adaptive response in the intestinal epithelium in numerous models of inflammatory bowel disease, such as colitis and T-cell induced diarrhea. The transcription factor family, hypoxia-inducible factor (HIF) has emerged as master regulators of the transcriptional response to hypoxic stress in normal and transformed cells. HIF heterodimers consist of an oxygen-labile α subunit (HIF-1α, HIF-2α) and a constitutively expressed HIF-1β subunit that mediate a wide spectrum of physiological and cellular adaptive responses, including angiogenesis, metabolic adaption, and erythropoiesis. HIF-1 has recently been implicated as a gut-protective factor in inflammatory bowel disease models by maintaining intestinal homeostasis. HIF-1 can also skew the differentiation of T cells to regulatory T cells (Treg) via the induction of FoxP3, thereby attenuating T-cell driven colitis. Although HIF-1 and HIF-2 share many overlapping functions, HIF-1 has been implicated in the inflammatory phenotype of M1 macrophages via inducible nitric oxide synthase (iNOS) whereas HIF-2 is involved in the anti-inflammatory phenotype of M2 macrophages via arginase-1 (Arg1). Based on these findings and that mucosal inflamed tissues are hypoxic, we hypothesized that the induction of the hypoxia-signaling pathway may limit GVHD-induced mucosal inflammation and injury.To determine the adaptive roles of HIF-1 and HIF-2 in GVHD, we first tested the major histocompatibility complex (MHC)-haploidentical C567BL/6 (B6,H2b) -> B6xDBA/2 (B6D2)F1 (H2b/d) model in which donor spenocytes (2x107) and anti-Thy1 + C' treated (T- cell depleted) bone marrow (ATBM) cells (5x106) are transplanted into B6D2F1 recipients after exposure to lethal irradiation (11Gy, split dose). B6 ATBM cells transplanted alone into B6D2F1 mice served as a negative control for all comparisons. Realtime PCR analysis demonstrated a modest increase in ileal mucosal HIF-2α expression 8 days (d) post-transplant (p<0.027). In contrast, HIF-1α mRNA levels were not induced. However, both HIF-1α and HIF-2α protein levels were upregulated 2-fold and 5-fold, respectively, in the ileal mucosa of B6D2F1 recipients receiving ATBM plus splenocytes, as determined by western blotting. Notably, Arg1 mRNA levels (HIF-2 target) were markedly upregulated during GVHD (p<0.018), whereas iNOS mRNA levels (HIF-1α target) were downregulated (p<0.01). Increased HIF-2α and Arg1 expression in the ileum as a consequence of GVHD was also observed in two MHC H2b-matched, minor histocompatability antigen (miHA)-mismatched models. BALB.B and CXB-2 mice were exposed to lethal irradiation (9Gy, split dose) and transplanted with B6 ATBM cells alone or along with host-presensitized B6 CD4+ T cells. After 8d, HIF-2α and HIF-1α mRNA ileal levels were increased and decreased, respectively, in both models undergoing GVHD. Similarly, Arg1 transcripts were increased by 12-fold (p<0.03) and 6.1 fold (p<0.007), respectively in B6->BALB.B and B6->CXB-2 models. However, after 20d post-transplant, a 4- and 3-fold decrease in Arg1 mRNA levels occurred in both models. Likewise, two anti-inflammatory, Treg-associated cytokines, interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) mRNA expression were elevated by 2-and 7-fold, respectively, after d8 in B6->BALB.B mice. TGFβ levels returned to baseline (p<0.05 vs d8) after 20d and IL-10 mRNA levels were reduced by 2.5 fold (p<0.029 vs d8). Lastly, in an ELISpot assay, the addition of a prolyl hydroxylase inhibitor, dimethyloxaloylgylcine (DMOG), a HIF activator, reduced the alloreactive interferon-γ response to vehicle levels (p>0.001) in a B6 anti-B6D2F1 mixed lymphocyte reaction.Taken together, our data suggest that HIF-2/Arg1 axis confers an anti-inflammatory response in the ileum after 8d of GVHD. However, after 20d, this response is inversely correlated with the lethality of the GVHD response. The amelioration of alloreactivity by DMOG suggests that the persistent activation of HIF may be necessary to dampen GVHD. Further studies will delineate the contribution of the HIF-2 response in the maintenance of intestinal homeostasis and limiting T cell alloreactivity. Disclosures:Zilberberg:Onyx Pharmaceuticals: Research Funding. Dziopa:Onyx Pharmaceuticals: Research Funding.

Incidence, Risk Factors and Clinical Outcome Of Leukemia Relapses Due To Loss Of The Mismatched HLA Haplotype After Partially-Incompatible Hematopoietic Stem Cell Transplantation

▪ IntroductionAllogeneic Hematopoietic Stem Cell Transplantation (HSCT) represents the best curative option for many patients with high-risk myeloid malignancies, mainly due to its potent immuno-mediated antileukemic effect. Still, post-transplantation relapse remains an unsolved issue. We and others described genomic loss of the mismatched HLA haplotype as a mechanism by which leukemic cells evade donor T cell-mediated immune pressure and cause clinical relapse after partially HLA-incompatible HSCT (Vago et al, N Engl JMed, 2009), but the actual incidence and risk factors of this phenomenon are to date largely unknown. MethodsWe analyzed retrospectively 224 consecutive partially HLA-mismatched HSCTs performed in our Institute in the last ten years (Unrelated Donor, UD: 60; Mismatched Related Donor, MMRD: 164) in patients affected by myeloid malignancies (Acute Myeloid Leukemia, AML: 173; Myelodisplastic Syndrome, MDS: 27, Myeloproliferative Neoplasms: 17; others: 7). All patients received myeloablative conditioning and infusion of donor T cells, either as part of the graft or as an add-back. Patients' follow-up included bone marrow genomic HLA typing to identify HLA loss relapses. In selected cases of HLA loss relapse cryopreserved serial serum samples harvested after HSCT were analyzed for the eventual presence of anti HLA Class I or Class II antibodies. ResultsWe documented 77 cases of relapse: 66 after MMRD and 11 after UD HSCT. Out of 77 relapses 21 (27%) were due to genomic loss of the mismatched HLA in leukemic cells. HLA loss occurred in 19 patients with AML, one with MDS and one with myelofibrosis. All the 21 cases of HLA loss occurred after MMRD HSCT (32%), so the analysis for putative risk factors were limited to this subgroup of transplants (n=164), comparing the frequencies of putative risk factors between patients with HLA loss and “classical” relapses (n=21 and 45, respectively). HLA loss relapses occurred significantly later than their classical counterparts (median time to relapse 307 vs 86 days, p<0.0001) in this very high-risk population, suggesting that outgrowth of the mutant variants require a considerable lapse of time. None of the disease-related factors we addressed (amongst which disease subtype, cytogenetics, molecular profile and disease status at HSCT) correlated significantly with eventual HLA loss. Use of an unmanipulated T cell-repleted graft resulted to be a risk factor for HLA loss relapses (Chi2=6.36; p=0.01). Both acute (HR:4.67, CI 95%: 1.53-14.22; p=0.007) and chronic (HR: 1.71; CI 95%: 0.68-4.28; p=0.01) Graft-versus-Host Disease (GvHD) occurred more frequently in patients with HLA loss relapses. Intriguingly, HLA-C*04 was more frequent in the mismatched haplotype of patients with HLA loss as compared to those with classical relapse (Chi2= 8.07; p=0.04), possibly suggesting an higher immunogenicity of the allele, hinted also by a similarly higher frequency in patients who did not relapse (Chi2=2.77; p=0.096). In our series, predicted NK alloreactivity had no apparent impact on eventual HLA loss. In none of the five patients studied to date we could evidence circulating anti-HLA antibodies, suggesting that humoral immunity does not play a major role in this phenomenon. Since lymphocyte infusions from the original donor are expected to be inefficacious to treat HLA loss relapses, whenever fit these patients were candidate to re-transplantation from alternative donors, HLA-mismatched and putatively alloreactive against the relapsed leukemia. Still, outcome was poor, with 5 of 8 re-transplanted patients dying of transplant-related mortality and only one alive in complete remission at a follow-up of 18 months. ConclusionsGenomic loss of the mismatched HLA haplotype is an extremely frequent mechanism of leukemia immune evasion and relapse after MMRD HSCT. It appears to be prompted by selective immune pressure mediated by donor-derived T cells, and accordingly occurs more frequently upon T cell-repleted transplants and in the presence of acute and chronic GvHD, clinical hallmarks of T cell alloreactivity. Conversely the role of NK and B cells in HLA loss needs further investigation, but appears to date less pronounced. Given the poor outcome of re-transplantation, mainly due to toxicity, novel diagnostic and therapeutic approaches are needed to anticipate the detection and improve the treatment of these frequent variants of leukemia relapse. Disclosures:Bordignon:MolMed SpA: Employment. Bonini:MolMed SpA: Consultancy.

Risk Factors For Steroid-Refractory Acute Graft-Versus-Host Disease After Allogeneic Stem Cell Transplantation From Matched Related Or Unrelated Donors

The standard risk factors for acute graft-versus-host disease (aGVHD) after allogeneic stem cell transplantation (allo-SCT) from related or unrelated donors are well defined and include HLA mismatch or unrelated donor, older recipient age, and female donor for male recipient (FM). The steroid-refractory (SR) forms of aGVHD are important to consider because they often have a major deleterious impact on transplant outcome. Unfortunately, the specific risk factors for SR aGVHD are less clearly defined. To characterize these risk factors after allo-SCT from matched related or unrelated donors, we undertook a retrospective analysis of adult patients transplanted at our center between 01/01/2000 and 12/31/2012. Steroid-refractory aGVHD was defined as aGVHD progressing after 3 days of treatment, or unchanged after 7 days, or in incomplete response after 14 days. GVHD occurring after donor lymphocytes infusion were excluded. Overall survival (OS) was calculated using the Kaplan-Meier estimate. Cumulative incidences (CI) were used for SR aGVHD in a competing risk setting with death as a competing event. The Gray test was used to compare CI curves. For multivariate analysis, the variables with p value &lt; 0.1 were entered into a Fine-Gray model and the least significant variables were excluded in sequential fashion until all remaining factors were significant at the p=0.05 level. The variables considered were recipient age (≥ vs &lt; 50 years, median age), female vs male donor, FM vs other combinations, matched related (MRD) vs matched unrelated donor (MUD), peripheral blood (PB) vs bone marrow (BM) graft, GVHD prophylaxis with cyclosporine (CsA)+metho vs others, myeloablative (MAC) vs reduced-intensity conditioning (RIC) regimen, antithymocyte globulin (ATG) vs no ATG, number of CD34+ cells in the graft ≥ vs &lt; 5.6 x 106/kg (median number), early (CR1, PR1, chronic phase, or untreated) vs advanced disease, CMV-seropositive vs seronegative recipient. Unrelated donors were matched at the allele level for HLA-A, B, C, DRB1, DQB1. Six hundred and thirty four patients were identified and included in the present study. The median age was 50 years (18-67). Diseases were AML (n=230), ALL (n=104), myeloma (n=80), NHL (n=74), Hodgkin's disease (n=18), MDS (n=47), CLL (n=29), CML (n=18), aplastic anemia (n=19), and MPS (n=15). Status at transplant were CR1 or PR1 or chronic phase (n=260), &gt; CR1 or PR1 (n=237), refractory (n=101), or untreated (n=36). Conditioning regimens were RIC (n=405) or MAC (n=229). Rabbit ATG was administered to 327 patients, of whom 298 received a RIC regimen. Donors were MRD (n=360) or MUD (n=274). Sources of stem cells were PB (n=452), BM (n=177), missing data (n=5). The prophylaxis of GVHD was CsA+metho for 339 patients. In the whole population, 71 patients presented a SR aGVHD at a median time of 29 days (8-137) after transplant, representing a CI of 11.2% ± 1.2%. Their OS at 1 year post-transplant was 27% ± 5%. In univariate analysis, the risk factors for SR aGVHD were MAC (p=0.02), MUD (p=0.02), no ATG (p=0.01), and a trend for FM (p=0.07). Other variables considered in univariate analysis were recipient age (p=0.6), female vs male donor (p=0.6), recipient CMV status (p=0.7), status at transplant (p=0.2), PB vs BM graft (p=0.9), number of CD34+ cells (p=0.4), and GVHD prophylaxis (p=0.6). In multivariate analysis, the risk factors for SR aGVHD were MUD (HR=2.5, 95%CI: 1.5-4.1, p=0.0003), FM (HR=2, 95%CI: 1.2-3.4, p=0.008), and no ATG (HR=2.1, 95%CI: 1.3-3.4, p=0.002). Patients were then divided into 3 groups. The CI of SR aGVHD was 2.7% ± 1.6% in the low-risk group (no MUD + no FM + ATG; n=112), 21.8% ± 3.4% in the high-risk group (MUD + FM +/- ATG, or MUD + no FM + no ATG; n=147), and 9.6% ± 1.5% in the intermediate-risk group (n=375); p=10-6. We conclude that MUD, FM, and no ATG were independent risk factors for SR aGVHD in adult patients after allo-SCT from MRD or MUD. These data support the prevalent roles of HLA and donor T cell alloreactivity in the pathogenesis of SR aGVHD and may help identify patients at high risk of SR aGVHD, candidates for experimental first-line therapies of aGVHD. Disclosures: No relevant conflicts of interest to declare.

Removal Of Alpha/Beta+ T Cells and Of CD19+ B Cells From The Graft Translates Into Rapid Engraftment, Absence Of Visceral Graft-Versus-Host Disease and Low Transplant-Related Mortality In Children With Acute Leukemia Given HLA-Haploidentical Hematopoietic Stem Cell Transplantation

T-cell depleted HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is a suitable option for patients in need of an allograft who lack a HLA-matched donor. Although it offers the advantage of being immediately applicable to virtually all patients, so far, graft manipulation with removal of all T lymphocyte subsets and of natural killer (NK) cells has been associated with an increased risk of life-threatening infections, as well as, in some studies, of leukemia recurrence.We recently developed a new method of graft manipulation based on the physical removal of αβ+ T cells and CD19+ B cells, which permits to leave mature NK cells and γδ+ T cells in the graft. We, thus, started a formal study (NCT01810120) in children with acute leukemia aimed at evaluating the safety and efficacy of this approach. As of April 2013, we enrolled 45 patients (pts; 29 males, 16 females). Median age at HSCT was 10.1 years (range 0.9-17.9). Thirty-five pts had acute lymphoblastic leukemia (ALL) and 10 acute myeloid leukemia (AML); all children were transplanted in morphological complete remission (CR). Fifteen pts were transplanted in first CR, 27 in second CR and 3 in more advanced CR. All pts transplanted in CR1 had either poor cytogenetic/molecular characteristics or high levels of minimal residual disease (MRD) at the end of induction therapy. The donor was either the mother (n=25) or the father (n=20); according to the model of KIR/KIR ligand disparity, 22 pts were transplanted from an NK-alloreactive donor. The median number of CD34+ cells, NK cells, γδ+ T cells, B cells and αβ+ T cells were 14.6, 31.7, 7.8, 0.08 and 0.04x106/kg, respectively. A myeloablative regimen, containing Total Body Irradiation in 34 cases, was given to all children, who also received anti-thymocyte globulin (12 mg/kg over 3 days, from -5 to -3). Rituximab (200 mg/m2) was administered on day -1 to further prevent EBV-related lymphoproliferative disorders. No pharmacological graft-versus-host disease (GVHD) prophylaxis was employed after transplantation.Sustained primary engraftment occurred in 44/45 pts, the remaining child being successfully re-transplanted from the other parent. The median time to reach an absolute neutrophil count >0.5x109/L and a platelet count >50x109/L was 13 days (9-18) and 11 days (8-20). No child developed gut or liver acute GVHD. Thirteen pts experienced skin-only grade I-II GVHD, this leading to a cumulative incidence (CI) of 29% (95% confidence interval, CO. IN., 18-45). Only 2 pts developed skin limited chronic GVHD. Two pts died for causes other than disease relapse (both in the first 60 days after HSCT), the CI of transplantation-related mortality (TRM) being 4% (CO. IN. 1-16). Seven pts relapsed, the CI of disease recurrence being 16% (CO. IN. 6-32). With a median follow-up of 11 months (range 2-30), the 2-year Kaplan Meier estimate of leukemia-free survival (LFS) was equal to 75% (CO. IN. 57-86); this value was 73% (CO. IN. 52-85) for pts with ALL. LFS of pts who did or did not experience skin-only acute GVHD was 83% (CO. IN. 48-96) and 72% (CO. IN. 50-86), p=NS. The probability of LFS of the 39 pts with either negative or low (<1x10-4) MRD at time of HSCT was 81% (CO. IN. 62-91) as compared to 25% (CO. IN. 1-67) of the 6 pts with positive MRD (p<0.01). The CI of cytomegalovirus (CMV) reactivation was 53% (CO. IN. 29-68). In the first 45 days after HSCT, circulating T cells mainly consisted of the γδ+ subset, while αβ+ T lymphocytes predominated thereafter. CMV reactivation promoted the expansion of Vδ1+ T cells. Mature NK cells expressing inhibitory KIRs and negative for NKG2A, including also the alloreactive cells, were detectable at 1 month in peripheral blood after HSCT, showing the persistence of transplanted cells with potential anti-leukemia activity. These percentages increased at later time points, reaching values comparable to those found in the HSCT donor.These data indicate that a selective graft manipulation results into effective prevention of both acute and chronic GVHD, rapid recovery of neutrophil and platelet counts and low TRM. Although the median observation time is still limited, also the CI of disease recurrence is encouraging. Post-transplant add-back of limited numbers of αβ+ T cells transduced with the iCasp9 suicide gene could optimize the recovery of this subset in early weeks after HSCT, maximizing immune-mediated viral and leukemia control, without an unacceptable risk of GVHD. Disclosures:No relevant conflicts of interest to declare.

The Histone Methyltransferase Ezh2 Is a Crucial Epigenetic Regulator Of Allogeneic T Cell Responses and Graft-Versus-Host Disease In Mice

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies, however, it remains to be limited by morbidity and mortality related to graft-versus-host disease (GVHD). During GVHD, donor T cells are activated by host antigen-presenting cells (APCs) and differentiate into effector cells that mediate host tissue damage. Development of alloreactive effector T cells requires orchestrated expression of myriad genes initiated by host APCs. However, little is known about the key epigenetic factor that controls this process. Histone methylation, which is catalyzed by histone methyltransferanses (HMT), has been correlated with genes encoding effector cytokines and transcription factors critical for effector differentiation. We recently demonstrated that inhibition of histone methylation using 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD through induction of apoptosis in alloreactive T cells. However, since DZNep reduced multiple histone methylation marks, including trimethylation of histone H3 at lysine 4 (H3K4me3), H3K27me3, H3K36me3 and H4K20me3, the key HMT that regulates the transcription program important for allogeneic T cell responses remains unclear. Using genetic approaches and mouse GVHD models, we identify that the HMT Ezh2, which specifically catalyzes H3K27me3 and acts primarily as a gene silencer, plays a central role in regulating allogeneic T cell proliferation, differentiation, and function during the GVHD process. Transfer of donor T cells derived from Ezh2 conditional knockout C57BL/6 (B6) mice did not induce GVHD in lethally irradiated allogeneic BALB/C mice, with all recipients surviving free of disease. In contrast, wild-type (WT) donor B6 T cells caused uniformly lethal GVHD in these recipients. We found that loss of Ezh2 selectively impaired alloantigen-specific T cell responses. Three days after allo-HSCT, both Ezh2-deficient and WT T cells were similarly activated to proliferate in vivo. However, 7 days after transplantation, there was a significant reduction in the number of highly proliferative Ezh2-deficient T cells compared to WT T cells, leading to a dramatic reduction of infiltrating Ezh2-deficient T cells in GVHD target organs. Interestingly, conditionally deleting Ezh2 did not affect proliferation and survival of B6 T cells that were transferred into lethally irradiated syngeneic B6/SJL mice by 7 days after transplantation. Thus, Ezh2 is critical for the continual proliferation of alloantigen-responding T cells during later stages of GVHD induction. In contrast, Ezh2 is dispensable for both the initial activation and proliferation of T cells upon alloantigen-priming and homeostatic expansion of T cells in response to lymphopenia. Previous studies suggest that IFN-g-producing alloreactive effector cells cause damage to the liver and gastrointestinal tract. We found that loss of Ezh2 led to selective reduction in frequency of IFN-γ-producing effector cells 7 days after allo-HSCT. This effect was accompanied with decreased expression of transcription factors TBX21 and STAT4, both of which are crucial for effector differentiation. These data suggest that in addition to regulating the transcription program critical for producing sufficient numbers of alloreactive effector T cells needed to mediate GVHD, Ezh2 is also important for orchestrating genes required to promote IFN-γ production in effector cells. Importantly, we found that Ezh2 ablation retained anti-leukemia activity in alloreactive T cells, leading to improved overall survival of transplant recipients. Collectively, these findings identify Ezh2 as a key epigenetic regulator that controls allogeneic T cell responses and GVHD. We propose that pharmacological modulation of Ezh2 using specific inhibitors should be investigated as a novel therapeutic strategy after allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Influence Of Killer Immunoglobulin-Like Receptor (KIR) and HLA Genotypes On Outcomes After Reduced-Intensity Conditioning Allogeneic Hematopoietic Stem Cell Transplantation For Patients With AML and MDS: A Report From The Center For International Blood and Marrow Transplant Research Immunobiology Working Committee

Disease relapse is a significant cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). In the setting of reduced-intensity conditioning (RIC), a graft-versus-leukemia (GVL) effect is critical for successful outcomes in patients with advanced myeloid malignancies. A GVL effect has been attributed in part to donor-derived alloreactive natural killer (NK) cells, which are regulated by interaction of KIRs with their HLA-class I ligands. Several models of NK reactivity (missing KIR ligand, centromeric haplotype-B content, non-tolerized KIR2DS1) have been associated with improved outcomes following HLA-matched, HLA-mismatched, related, and unrelated donor HCT, particularly for AML patients given myeloablative conditioning. The effect of KIR-HLA combinations on outcomes after RIC HCT, however, is not known. We retrospectively analyzed donor-recipient KIR/HLA genotypes from 929 URD HCTs facilitated by the National Marrow Donor Program for patients with AML (n=624) or MDS (n=305) treated with RIC between 1990 and 2007. 664 donor-recipient pairs were 10/10 HLA-matched and 265 were 9/10 HLA-matched. 332 (37%) patients received ATG and 73 (8%) received alemtuzumab. P-values less than 0.01 were considered significant. We hypothesized that donor-recipient KIR-HLA interactions may be associated with improved post-transplant outcomes following RIC HCT for AML and MDS. Patients lacking the HLA-Bw4 ligand for donor inhibitory KIR3DL1 experienced lower relapse following HCT at 1 year (27 vs 36%, p=0.002) and 5 years (31 vs 42%, p=0.003) compared to patients with the Bw4 ligand (Figure). The lower risk for relapse was confirmed in multivariate analysis (HR 0.71, p=0.005). However, there was no significant association of Bw4 ligand with disease-free survival (HR 0.84, p=0.05) and overall survival (HR=0.97, p=0.70). Risk for acute GVHD was higher among patients lacking KIR ligands after adjusting for other clinical factors. In particular, patients lacking HLA-C2 for donor KIR2DL1 experienced higher grade 2-4 (HR 1.3, p=0.005) and 3-4 acute GVHD (HR 1.5, p=0.002), and patients lacking multiple KIR ligands experienced higher grade 3-4 acute GVHD (HR 1.5, p=0.007). The analysis was then restricted to AML patients, the patient population with greatest reported KIR-HLA effects. Patients whose donors were KIR2DS1+ and HLA-C2C2 (n=33) had higher transplant-related mortality (TRM) (HR, 2.4, p=0.002) compared to all other patients. There was no significant effect of KIR2DS1 with HLA on relapse. In a multivariate analysis, lack of HLA-C2 in AML patients was associated with higher grade 2-4 (HR 1.4, p=0.002) and 3-4 acute GVHD (HR 1.5, p=0.01), and risk for grade 3-4 acute GVHD was higher in patients lacking multiple KIR ligands (HR 1.6, p=0.005). There were no significant associations between donor homozygosity for the centromeric B-haplotype (cenBB) or overall B-haplotype KIR content and RIC HCT outcomes.Overall, these results suggest that in the RIC HCT setting, lack of Bw4 ligand for KIR3DL1 is associated with a lower risk of relapse for AML/MDS. This observation corroborates previous findings in myeloablative HCT transplants. Furthermore, tolerance of donor KIR2DS1 by HLA-C2C2 was associated with worse outcome, as manifested by higher TRM, in AML patients. In contrast, it appears that in RIC HCT homozygosity for the centromeric B-haplotype does not have a significant role in leukemia relapse. The associations of KIR ligands with acute GVHD were not previously observed suggesting that NK cell alloreactivity depends on multiple variables, including RIC. Further elucidation of the biology of NK cell alloreactivity in the RIC setting may provide guidance for future approaches to help optimize conditions for generating GVL reactions without GVHD and less TRM in this transplant population. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

NK Cell KIR Ligand Mismatches Influence Engraftment Following Combined Haploidentical and Umbilical Cord Blood (UCB) Transplantation In Patients With Severe Aplastic Anemia (SAA)

BackgroundAllogeneic hematopoietic stem cell transplant (HSCT) is curative for patients (pts) with severe aplastic anemia (SAA). For SAA pts who lack HLA-identical donors, we explored a HSCT approach that co-infuses PBSCs enriched for CD34+ cells from a haplo-identical relative combined with a single umbilical cord blood unit (UCB). Although most pts undergoing this approach had early haplo-myeloid engraftment that was eventually supplanted by the UCB unit, engraftment patterns were highly variable; in some pts full cord myeloid chimerism was delayed greater than one year, while others had loss of cord engraftment with sustained full haplo-donor myeloid chimerism. Here, we investigated the impact of various patient, UCB, and haplo-donor characteristics, as well as NK cell KIR ligand mismatches between graft sources, on engraftment kinetics following haplo-cord HSCT. MethodsPts with SAA or SAA evolved to MDS unresponsive to immunosuppressive therapy with severe neutropenia (ANC<500), who lacked an HLA-matched donor and had an available haploidentical family member, and had at least one ≥ 4/6 matched UCB unit with a minimum TNC dose ≥1.5x107 cells/kg were eligible for HSCT. Pts were conditioned with cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine-ATG (160 mg/kg) and 200 cGy of total body irradiation. On Day 0, pts received a CD34 selected (Miltenyi CliniMacs) G-CSF mobilized haplo-donor allograft combined with a single UCB. Tacrolimus and MMF were given for GVHD prophylaxis. Results16 pts (median age: 18.9 yrs, range: 4.5-27.9) including 13 SAA and 3 with SAA evolved to MDS underwent haplo-cord transplant. Ten pts received a 4/6, and six pts received a 5/6 HLA-matched UCB unit. A median 3.6x107 TNC/kg (range: 1.96-6.93) from the UCB unit, and 3.3x106CD34+ cells/kg (range: 3.0-4.1) from the haplo-donor were transplanted. All 16 pts had sustained engraftment with 15/15 pts evaluable past day 100 having transfusion independence. At a median follow-up of 570 days (range: 55-1826), 14/16 pts survive for an overall survival rate of 81.8%. Two pts died at day 414 and 402 post-HSCT from viral related complications (CMV pneumonitis and limbic encephalopathy). Neutrophil and platelet recovery occurred at a median 10 (range: 9-22), and 21 (range: 10-213) days post-HSCT, respectively. By post-HSCT day 11, 15/16 pts had neutrophil recovery. Cord myeloid engraftment (cord ANC>500, calculated from chimerism data) occurred in 13/16 pts at a median 42 days. 3/16 did not achieve a cord ANC>500 but had sustained haplo-donor engraftment. The cumulative incidence of acute grade II-IV GvHD was 38.1%. Engraftment profiles were highly variable among pts; 12 achieved full cord chimerism in all cell lineages, 2 remained mixed haplo-cord chimeras, and 2 failed to have UCB engraftment but had sustained 100% haplo-donor myeloid chimerism. Higher degrees of HLA matching (out of 10 alleles) between recipient and UCB unit were associated with faster rates of full cord engraftment (p=0.006) and a higher probability of complete loss of haplo-donor chimerism (p=0.018). KIR ligand incompatibility in the haplo vs. cord direction (defined as the presence of a KIR ligand in the haplo-donor graft that is absent in the UCB unit at HLA epitopes Bw4, HLA-C Group 1 & 2, HLA-A3, and HLA-A11) negatively impacted cord myeloid engraftment. 5/5 (100%) pts who failed to achieve full cord myeloid chimerism by post-HSCT day 400 had haplo vs. cord KIR ligand incompatibility. Moreover, both pts who failed to have UCB engraftment and had sustained haplo-donor chimerism had haplo vs. cord KIR ligand incompatibility. In contrast, only 3/11 (27%) pts who achieved full cord myeloid chimerism post-HSCT by day 231 had haplo vs. cord KIR ligand incompatibility (p=0.026). KIR ligand incompatibility in the cord vs. haplo direction showed no significant effect on haplo-donor myeloid engraftment. ConclusionThese results show that haplo-cord HSCT is an effective treatment option for pts with SAA who lack an HLA-matched donor. Further, these results suggest that NK cell alloreactivity, occurring as a consequence of KIR ligand mismatch between the two graft sources, may have a negative impact on cord engraftment when haplo vs. cord KIR ligand incompatibility is present. In summary, this study highlights a novel factor that should be considered during graft selection for haplo-cord transplantation. Disclosures:No relevant conflicts of interest to declare.

Recruitment Of T Cells and Macrophages To The Eyes In Recipients Of Allogeneic Hematopoietic Stem Cell Transplants Correlate With Inflammatory Cytokine Presence In Ocular Gvhd

Allogeneic hematopoietic stem cell transplantation (HSCT) has become the standard of care for the treatment of several life-threatening hematologic malignancies as well as certain immunodeficiency disorders. Unfortunately, as the survival rate of patients with these diseases is improved, the quality of life is negatively impacted by the development of Graft vs. Host Disease (GVHD). GVHD is a complex, multi-organ disorder arising from an immunological attack by donor allo-reactive T cells that results in damage to vital organs including the liver, skin, hematopoietic compartment and the ocular surface of the eye. Ocular GVHD occurs in >60% of these patients and is characterized by dry eye, conjunctiva damage, punctate keratopathy, corneal ulceration and perforation. Despite the high frequency of eye involvement in patients undergoing GVHD, little is known regarding the underlying immune mechanisms responsible for ocular GVHD, limiting the ophthalmic care of these patients to palliative therapies and global anti-inflammatory drugs. In this study, we examined the ocular and immunological changes occurring in recipients of MHC-matched, minor antigen mis-matched donor HSCT. C3H.SW (H-2b, Ly9.1+) mice transplanted with EGFP+ B6 (H-2b, Ly9.1-) T cell depleted bone marrow cells (TCD-BM) supplemented with T cells: a) underwent weight loss and began exhibiting clinical signs of GVHD ∼3wks post-HSCT, b) contained damaged thymuses, c) expressed an inverted CD4/CD8 ratio in the peripheral lymphoid compartments, d) contained activated effector cells and e) low CD19 levels. Importantly, these mice also developed ocular surface disease evidenced by progression of ocular surface damage characterized by increased corneal fluorescein staining and ulceration by week 6 (Figure). Furthermore, histological analyses demonstrated that only mice that developed systemic GVHD exhibited corneal thickening and epithelial irregularity. Ocular pathology was also associated with conjunctiva involvement indicated by significant goblet cell destruction as well as dense inflammatory cell infiltrates identified by intra-vital fluorescent microscopy (Figure). IHC and flow analyses demonstrated donor EGFP+ Ly9.1- CD4+ and CD8+ T cells. Notably, significant levels of Ly9.1- EGFP-CD11b+ macrophages had also infiltrated the ocular surface. In contrast to the systemic CD8>CD4 GVHD associated phenotype, the T cell infiltrate in the ocular compartment was reversed; i.e. CD8<CD4. We detected IFNγ and TNFα mRNA from corneal tissue, which is consistent with Th1 effector allo-reactive cells and M1 inflammatory macrophages involvement in ocular GVHD. In total, the present findings have identified alterations and pathology in the eye and adnexa reflective of ocular GVHD and unequivocally demonstrate the presence of donor T cells in the ocular surface. We hypothesize that T cell–macrophage interactions underlie the pathology detected in this pre-clinical model and studies are underway to develop local therapeutic modalities targeting these infiltrative populations. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

An Anti-CD154 Domain Antibody Prolongs Graft Survival and Induces Foxp3+ iTreg in the Absence and Presence of CTLA-4 Ig

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc-mediated deletion of CD154-expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti-CD154-induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti-CD154 therapy, two alternative means of targeting the CD40–CD154 pathway were used: a nonagonistic anti-CD40 antibody and an Fc-silent anti-CD154 domain antibody. We compared these therapies to an Fc-intact anti-CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti-CD40 mAbs as well as Fc-silent anti-CD154 domain antibodies were equivalent to Fc-intact anti-CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen-specific T cells and promote the conversion of Foxp3+ iTreg. Importantly, iTreg conversion observed with Fc-silent anti-CD154 domain antibodies was preserved in the presence of CTLA4-Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

Donor Selection for Killer Immunoglobulin-like Receptors B Haplotype of the Centromeric Motifs Can Improve the Outcome after HLA-Identical Sibling Hematopoietic Stem Cell Transplantation

After hematopoietic stem cell transplantation (HSCT), natural killer (NK) cell alloreactivity in HLA cells of recipients is regulated by killer immunoglobulin-like receptors (KIRs) on donor NK cells. The effect of KIRs on HSCT outcomes is controversial, particularly in those undergoing HLA-identical sibling HSCT. In this study, effects of KIR and HLA genotypes on the HSCT outcome were investigated in a 5-year retrospective study comprising 219 patient-donor pairs undergoing HLA-identical sibling HSCT for myeloid and lymphoid malignancies. We found that 39.7% (87 of 219) of these pairs, which were KIR mismatched, had better overall survival (OS) and reduced grade III to IV acute graft-versus-host disease (aGVHD), especially in acute myeloid leukemia (AML) patients. Bx1 donor KIR genotype with haplotype B on a telomeric region was a risk factor for the OS and relapse-free survival (RFS). Donor centromeric (c) and telomeric (t) KIR haplotype analysis showed that donor KIR cB-tA/tB was associated with improved OS and RFS compared with cA-tA or cA-tB. Furthermore, donor KIR B haplotype of the centromeric motifs (Cen-B) was an independent beneficial factor in improving OS and RFS and in protecting from relapse after HSCT. In AML patients, the occurrence of aGVHD was significantly lower in HLA-C1 group compared with that in HLA-C2 group, although such effect was not observed in patients with acute lymphoblastic leukemia or chronic myelogenous leukemia. Our results suggest that KIR could impact outcome and donor KIR haplotype with Cen-B confer significant survival benefits to HLA-identical sibling HSCT.

13-OR

For hematopoietic stem cell transplantation (HSCT) HLA identical sibling donors or HLA matched unrelated donors are the most ideal donors. As 10/10 (HLA-A, -B, -C,-DRB1, -DQB1) matched donors, are not available for all patients, the identification of permissive/non-immunogenic mismatches is important to inmprove the outcome of HLA mismatched transplants. Alleles that share the amino acid sequence of the antigen recognition site (ARS)(exon 2 and 3 for HLA class I or exon 2 for HLA-DRB1) or differences outside the peptide binding groove or α -helices (as the HLA-C*03:03 vs C*03:04 mismatch) are good candidates for permissive mismatches as these differences are not likely to induce T cell alloreactivity. An inventory of the functional impact of such mismatches was performed by in vitro cellular assays including the Cytotoxic T Lymphocyte precursor test (CTLp) for HLA-class I allele differences and the MLC, IFN- γ elispot and Primed Lymphocyte Test (PLT) for HLA-DRB1 differences. CTLp assays were performed for: HLA-A*23:17 vs 23:01 (differ at pos 283, exon 5), HLA-C*07:06 vs *07:01 (pos 307 & 324 exon 5 & 6), HLA-C*04:30 vs 04:01 (pos 327, exon 7), HLA-C*03:62 vs 03:03:01 (pos 197, exon 4), HLA-C*03:03 vs 03:04 (pos 91, exon 3) and HLA-B*35:02 vs 35:04 (pos 109, exon 3). The only positive CTLp test was for HLA-C*07:06 vs *07:01. DRB1*14:01 vs DRB1*14:54 differ at pos 112 and a 2nd mismatch of DRB3*02:01 vs DRB3*02:02, respectively, yielded complicated results. Five of 6 combinations tested did not respond in the MLC and 3 of 6 in the Elispot against the combined DRB1/DRB3 mismatch and only in one direction; DRB1*14:01/DRB3*02:01 against DRB1*14:54/DRB3*02:02. PLT results suggested that the DRB1* mismatch was responsible for the response. In conclusion, mismatches involving positions outside the ARS are not very immunogenic. However, some mismatches can lead to T cell reactivity in vitro. The impact of these mismatches on clinical outcome of HSCT remains to be established.

Immunosuppression withdrawal following liver transplantation: The older, the wiser… but maybe too late

Immunosuppression withdrawal following liver transplantation: The older, the wiser… but maybe too late