ALBERT

Description: Background: Although much is known about the structure and immunogenicity of red blood cell (RBC) antigens, little is known about their processing and presentation by antigen presenting cells (APC). Red blood cells are a unique immunogen, in that they are given intravenously, without inflammation, and typically don’t enter peripheral tissues and lymphatics. Unlike pathogens, which cause an immune response in the majority of patients, only a small minority of chronically transfused patients develop alloantibodies to RBC antigens. In a murine model of RBC transfusion, we have previously reported that recipient inflammation, induced by Poly (I:C) (a double-stranded RNA that mimics viral inflammation), significantly enhances alloimmunization to RBC antigens. In this report, we explore the role of antigen presenting cells in the immune response to antigens on transfused RBCs, in an uninflammed state as well as in the presence of Poly (I:C). Methods: 3, 3-dihexadecyloxacarbocyanine perchlorate (DiO) was used as a fluorescent RBC label. Labeled RBCs were transfused into C57BL/6 recipient mice, in the absence or presence of inflammation with poly (I:C). 24 hours post-transfusion, APCs were analyzed in the spleen, liver, and lymph nodes. Macrophages (F4/80+) and dendritic cells (DC) (CD11c+) were gated on by flow cytometry, as were T cells (CD3+), B cells (CD19+) and RBCs (Terr 119+). RBC consumption was assessed by measuring DiO fluorescence in these cell populations. Results: In the absence of inflammation, the majority of RBCs are consumed by macrophages in the spleen, with 3 fold less consumption by liver macrophages and no consumption by lymph node macrophages. Both splenic and liver DCs consume 3 fold fewer RBCs than splenic macrophages. Recipient inflammation with Poly (I:C) alters this pattern, with a significant increase in consumption by both splenic and liver DCs and a decrease in consumption by splenic macrophages. As a negative control, no RBC consumption was seen after gating on non-phagocytic T cells or B cells. Likewise, measures of RBC consumption were not an artifact of RBC sticking to the APC surface, as staining for TER119 was negative. Discussion: Red blood cells are a unique immunogen, in that they circulate for many days, don’t enter lymphatics, and often don’t cause a detectable alloantibody response. These studies demonstrate that recipient inflammation with Poly (I:C), which we have previously reported enhances alloimmunization to transfused RBCs, significantly increases DC consumption of transfused RBCs. As DCs are typically considered to be more potent APCs than macrophages, and as we have previously shown that Poly (I:C) signficantly induces co-stimulatory molecule expression on DCs, these findings provide one potential mechanism by which inflammation enhances RBC alloimmunization. Ongoing studies are directly assessing the relative potency of these different APCs in their ability to activate CD4+ T cells specific for RBC antigens.

Description: CMV infection is reported to increase the incidence and severity of chronic GvHD and clinical data have shown that preemptive antiviral therapy decreased the risk of extensive chronic GvHD. Using mouse model of Allogeneic BMT, we investigated the mechanism for the association of MCMV infection and GvHD. We hypothesize that MCMV infection leads to generalized immune activation and increases the number of donor derived allo-reactive T cells and GvHD activity. Methods: A parenteral to F1 mouse BMT model was used to study anti-CMV immunity and GvHD. Low dose splenocytes (3x106) from MCMV immunized C57BL/6 donors (H-2b, Thy1.2+, CD45.1+) were transplanted with 5x106 T cell depleted bone marrow (TCD BM) from congeneic mice (H-2b, Thy1.1+, CD45.2+) into lethally irradiated (11Gy) CB6F1 recipients (C57BL/6 x Balb/C, H-2b/d, Thy1.2+, CD45.2+). Previous work has established this as a dose that protects against CMV without immediate lethality from GvHD. Non-GvHD control mice received a dose of Amotosalen treated splenocytes (10x106) and 5x106 TCD BM that protects against CMV without GvHD. Recipient mice were infected i.p. with a supralethal dose (2.5x104 pfu) of MCMV 7 days post transplant. Clinical GvHD was monitored twice weekly by weight loss, hair loss, ruffled fur, diarrhea, and decreased activity. T cell chimerism in recipient spleen and thymus, and MCMV peptide specific tetramer+CD8+ T cells were determined by flow cytometry. Liver and lung viral loads were determined by counting PFU in tissue homogenates plated onto 3T3 confluent monolayers. Results: During the acute phase of MCMV infection (day 3 to 14 post infection), recipient mice that received 3x106 untreated donor splenocytes developed GvHD characterized by weight loss and higher mortality than the non-GvHD control mice. Although both GvHD+ and control mice effectively cleared the virus from their liver, delayed viral clearance from the lung was found in non-GvHD recipients. Viral clearance was associated with expansion of donor spleen-derived MCMV peptide specific tetramer+ CD8+ T cells. The kinetics of donor T-cell expansion varied significantly between the two treatment groups, with GvHD+ recipients showed rapid early expansion of donor derived T-cells followed by the development of GvHD with subsequent lymphopenia. Recipients of Amotosalen-treated splenocytes had more gradual expansion of total and 400-fold expansion of antigen specific T-cells with sustained lymphoid reconstitution. GvHD+ recipients of untreated splenocytes had complete chimerism comprised of 〉80% of CD8+ donor T cells whereas non-GvHD controls had significant expansion of host derived T cells following MCMV infection and lacked allo-reactive of donor- spleen-derived T cells. Thymic function was inhibited among animals that developed GvHD and preserved among control mice throughout the infectious phase. Very delayed CMV infection (on day 60 after transplant) in mice with established chronic GvHD exacerbated GvHD and was associated with delayed lung viral clearance. Conclusion: After CMV infection there is extensive expansion of allo-reactive T cells in GvHD+ mice with associated damage to the microenvironment in the spleen and thymus. Amelioration of the immuno-suppressive effect of CMV infection (in clinical transplantation) will likely require more effective prophylaxis and treatment of GvHD in allotransplant recipients.

Description: Background: Acute graft-versus-host disease (aGvHD) remains a critical barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nearly all patients who undergo allo-HSCT receive red blood cell (RBC) transfusions during the first 30 days post-transplant, but the long-term effects of transfusions on post-transplant outcomes remain unclear. Preclinical and clinical studies indicate that RBC transfusions can activate dendritic cells (DCs) sensitizing transplant recipients to minor histocompatibility antigens (miHA). Initial interactions between donor T cell and DC regulate donor T cell activation and proliferation. We hypothesized that RBC transfusion may contribute to aGvHD in allo-HSCT patients by activating DC and stimulating allo-reactive T cells or other inflammatory pathways. Methods: We conducted a retrospective study of 336 adult allo-HSCT patients at Emory University Hospital from 2007 to 2013. In cases involving multiple transplants, only data relevant to the first allo-HSCT was included. Graft sources were restricted to bone marrow and G-CSF mobilized peripheral blood (excluded cord blood and T cell depleted grafts). 181 patients (54%) were male and 155 (46%) were female. Patients received transplants from matched related donors (n=123, 37%) or matched unrelated donors (n=213, 63%) for treatment of AML (n=132), ALL (n=40), acute leukemia (n=5), MDS (n=41), CML (n=22), CLL (n=15), HD (n=6), NHL (n=33), AA (n=10), MM (n=7) or other diseases (n=25). aGvHD with onset of up to 150 days after allo-HSCT was the primary end-point. Leuko-reduced and irradiated RBC transfusions administered during the week prior to transplant and 30 days post-transplant (while patients were closely monitored at the transplant center) were provided by a single Blood Bank. The median follow up time was 14.6 months post transplantation (range, 0.3 to 65.1 months). The relationship of RBC transfusions to aGvHD was determined as a time-dependent variable or as a function of the total number of RBC units transfused. Results: 306 patients (91%) received RBC transfusions during the observation timeframe, while 30 (9%) did not require transfusion (median 6). 221 patients (66%) developed grade I-IV aGvHD with a distribution of 85 (25%), 72 (21%), 47 (14%), and 11 (3%) of patients with maximum grade of I, II, III and IV, respectively, while 115 (34%) patients did not show any signs of aGvHD. We compared transfusion history, prior to the diagnosis of GvHD, in patients who developed grade 0-II aGvHD (n=272, 81%) vs. grade III-IV aGvHD (n=64, 19%). Patients with grade III-IV aGvHD received more RBC units (median 8) than patients with grade 0-II aGvHD (median 4). In univariable Cox regression analysis, factors significantly associated with grade III-IV acute GvHD were HLA match (HR 2.22, p=0.004), number of RBC units per week (HR=1.26, p

Description: Abstract 3348 Background: Technologies have recently been developed for rapid determination of extended human erythrocyte antigen (xHEA) phenotypes. For example, a semi-automated method using allele-specific oligonucleotides targeted against 32 clinically significant minor RBC antigens has been used to determine donor xHEA phenotypes from whole blood samples. This approach is currently used by blood collection centers and medical centers with blood collection facilities (both sites have access to linked donor whole blood samples). Broader access to xHEA information closer to the point-of-care (e.g. Transfusion Services at a Medical Center without a blood collection facility) may provide an opportunity to enhance patient care by more quickly and broadly providing units with xHEA phenotypes (Klapper et al., 2010.) However, transfusion services would need to use integrally attached segments for testing, and with leukoreduced (LR) RBC units these segments have very low numbers of white blood cells (WBC) (and therefore DNA), potentially limiting analysis. This study was performed to determine whether a HEA-elongation mediated multiplex assay in solution (HEA-eMAP-S) (Xin et al., 2010) could accurately genotype segments from LR-RBC units for 32 clinically significant minor RBC antigens. Methods: Segments from pre-storage LR-RBC units (American Red Cross), 〈 14 days old, were obtained from a large tertiary care Children's Hospital in the Southeastern US and residual WBC were quantified by flow cytometry. DNA was extracted using an extraction method developed at BioArray SolutionS (BAS) using commercial reagents (Qiagen, Inc., Valencia, CA), and then amplified with the Universal Beadchip™ package (HEA LR-eMAP-S Beadchip™ Kits) which contains allele specific oligonucleotides directed to 32 clinically significant blood group antigens (c, C, e, E, V, VS, K, k, Kpa, Kpa, Jsa, Jsb, Jka, Jkb, Fya, Fyb, M, N, S, s, Lua, Lub, Dia, Dib, Coa, Cob, Doa, Dob, Joa, Hy, Yta, Ytb mutation for hemoglobin S). DNA analysis results were correlated with RBC storage solution, WBC filter type, and serologic minor RBC antigen phenotypes of the units. Results: 102 LR-RBC units from whole blood donations were studied, 74 /102 (73 %) stored in AS-1 and 28 /103 (27 %) in CPDA-1 solution. All AS-1 units were pre-storage LR with Fenwal Sepacell Flex Excel Filters and all CPDA-1 units were pre-storage LR with Whole Blood Fenwal Filters (Fenwal Inc. Lake Zurich, IL). All units demonstrated 〈 5 × 106 WBC/unit with 47 % having 〈 4 × 104 WBC/unit, which is at or below the limit of flow cytometric detection. Complete genotyping data was obtained from all samples. Ten samples showing initial indeterminate results on Diego and one for N antigens produced complete results after repeat testing. Fifty-four percent of units were serologically phenotyped for 1–8 antigens by the blood collection center; there was 100% correlation between predicted phenotype from DNA analysis and serology for these units. Conclusions: The HEA LR-eMAP-S DNA analysis can be applied to optimally pre-storage LR-RBC units yielding 〉 99 % accuracy for all minor red blood cell antigens tested. The ability to perform this type of testing in a hospital transfusion service opens up new possibilities for transfusion services to select from their existing inventory and more efficiently allocate units to recipients with specific phenotypic requirements for RBC units. Disclosures: Josephson: Immucor: Speakers Bureau. DeMezzo:Immucor: Employment. Tanzi:Immucor: Employment. Enriquez:Immucor: Employment. Lin:Immucor: Employment. Hashmi:Immucor: Employment.

Description: Abstract 3561 Poster Board III-498 Background Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Immunosuppressive drugs limit clinical GvHD but increase relapse and susceptibility to opportunistic infections and also result in drug related toxicities. To develop an alternative approach to control GvHD, we tested the immunomodulatory immune properties of flagellin, a bacterial protein that agonistically binds with TLR5 and protects mice from radiation-induced gut injury, in murine allo-BMT models. Methods We used established BA.B10 (H-2K) → C57BL/6 (H-2b) MHC mismatched experimental models of allogeneic HSCT in which GvHD is a major complication. 50 μg LPS-free purified flagellin in PBS or PBS alone were administered intraperitoneally in two doses: 3 hours before fractionated irradiation (5.5Gy X 2 fractions) and 1 day post-transplant. Allografts were performed 1 day after irradiation and contained 5 million (M) T-cell depleted bone marrow (BM) cells and 5 M plastic non-adherent splenocytes from naïve BA.B10 donors. The primary end-points was survival; HSCT recipients were monitored twice a day for mortality and GvHD signs and recipients having more than 25% weight loss were sacrificed. Blood, spleen, thymus and BM were collected from surviving mice on day 132 post transplant, live cells counted, and immune phenotypes were analyzed by FACS. The numbers and phenotype of immune cells in organs from flagellin-treated HSCT recipients were compared to the similar immune cells per organs analyzed from a normal B6 mouse having similar age of HSCT recipients. Results Flagellin treated recipients had 15% weight-loss and 33% transplant-related death by 132 days post transplant versus severe acute GvHD and 100% early post-transplant mortality among control HSCT recipients that received PBS. Flagellin-treated recipients had 100% donor chimerism with limited clinical signs of GvHD. While total cell numbers per spleen (8.2 ± 5.4M) and thymus (7.1 ± 4.9M) were very low in flagellin-treated recipients compared to normal B6 mice (〉100M/organ), the cell numbers isolated from blood (8.9 ± 2.6 M/ml) and BM (104.5 ± 37.4 M) were similar to non-transplanted B6 mice (11.4M/ml blood and 108 M/BM, respectively). BM of flagellin-treated HSCT recipients contained similar numbers of CD4+ T cells (4.6 ± 2.7 M) and CD8+ T cells (2.5 ± 1.4 M) as normal B6 mice (4.03M and 1.3M, respectively). Numbers of naïve and memory CD4+ T-cells in the BM were similar between flagellin-treated and control mice: CD4+CD62L+(0.7 ± 0.2 versus 0.5M); CD4+CD62L- (3.9 ± 2.5 versus 3.5 M); CD4+ CD44hi (2.8 ± 1.4 versus B6 3.6M); and CD44lo (1.7 ± 1.3 M versus 0.44M). In contrast, flagellin-treated HSCT recipients had more naïve CD8+ T-cells but similar memory CD8+ T-cells in their BM compared with control mice: CD8+CD62L+(2.6 ± 1.4 versus 1.0M); CD8+CD62L- (1.7 ± 1.2M versus 0.3 M); CD8+CD44hi(0.8 ± 1.1 versus 0.7M); and CD8+CD44lo (0.7 ± 0.3M versus 0.6 M). The numbers of total CD3+ T cells, NK cells, and lin-CD11b-Sca-1+Ckit+ Stem cells in the BM were also similar comparing flagellin-treated recipients with non-transplanted B6 control mice. The number of CD3-B220+ B cells in the BM were lower in flagellin-treated recipients compared to B6 mouse (18.1 ± 3.2M versus 43.1M) as were the numbers of T-cells and B-cells per mL blood of flagellin-treated mice were found lower compared with the blood of normal B6 mouse: 0.8 ± 0.2M T-cells/mL versus 2.1M/mL; 5.5 ± 2.5M B cells/mL versus 9.1M/mL. Although the cellularity of the thymus in flagellin-treated animals was very low compared to normal B6 mice, a usual percentage (62.5 ± 10.5%) of thymocytes were of CD4/CD8 double positive, indicating functional thymopoiesis in these recipients. Conclusion Flagellin protected allogeneic HSCT recipients from irradiation-induced BM damage and prevented lethal GvHD in a major MHC mis-matched model of GvHD. Flagellin and other TLR5 agonists may be novel therapeutic approaches to prevent or reduce GvHD in allogeneic HSCT recipients. Disclosures: No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic stem cell transplantation (HSCT) can eradicate chemorefractory leukemia through the graft-versus-leukemia (GVL) activity of donor T cells. However, the clinical success of allo-HSCT is limited by the graft-versus-host disease (GVHD) activity of donor T cells. We have reported previously that donor bone marrow precursors of plasmacytoid dendritic cells (pre-pDCs) can activate donor T cells toward T-helper 1 immune polarization in murine allogeneic HSCT. To optimize the GVL activity of these activated donor T cells and limit their graft versus host activity, we engineered the cellular constituents of an allogeneic hematopoietic stem cell graft with highly purified hematopoietic stem cells, T cells, and pre-pDCs and studied their GVL and GVHD activities in a murine model of allogeneic HSCT. Transplanted donor pre-pDCs expanded in vivo for 2 weeks after transplant, and they markedly augmented the activation and GVL activity of donor T cells while attenuating their GVHD activity, leading to an improved therapeutic index. Bidirectional signaling between donor T cells and donor pDCs with IFN-γ synthesis by donor T cells inducing indoleamine 2,3-dioxygenase synthesis by donor pDCs limited GVHD by altering the balance between donor T-reg and inflammatory T cells. Manipulating the content of donor DC precursors in allogeneic HSCT is a novel method to optimize the balance between GVL and GVHD.

Description: Abstract 1465 Background: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Using flagellin, a bacterial protein that agonistically binds with TLR5, we previously reported that two doses of flagellin (before irradiation and after allogeneic HSCT with 5×106 splenocytes) in H-2K à H-2b model significantly reduced GvHD and had 67% long-term survival by 132 days post-transplant whereas control recipients had severe acute GvHD and 100% early mortality within 15 days post transplant. Here, we report the mechanism by which flagellin reduces GvHD using the same H-2K à H-2b HSCT murine model. Methods: 3×106 splenocytes and 5 ×106 T cell depleted bone marrow (BM) cells harvested from the congeneic H-2K donors were transplanted into lethally irradiated (11Gy) C57BL/6 (H-2b) recipients. 50-μg HPLC purified LPS-free flagellin diluted in ice-cold PBS were administered i.p 3 hours before irradiation and 24 hours after transplant. Control recipients were treated with PBS. Recipients (6/group) were sacrificed on day 4 and 10 after post transplant. Serum was collected to determine cytokines by ELISA and splenocytes were analyzed by FACS to determine immune cells phenotypes. Results: Although both Fla and PBS-treated recipients showed identical weight-loss within day 10 post transplant, surprisingly significantly lower numbers of cells/spleen were determined in the spleens of Fla recipients compared to control recipients [Fla 0.9 ± 0.1 (x106) vs PBS 1.7 ± 0.4(x106), p=0.002] on day 4 post transplant but not on day 10 [Fla 186.1 ± 35.1 (x106) vs PBS 151.2 ± 40.5(x106), p=0.43] post-transplant. We investigated the paradoxical immune response of flagellin on donor T cells on day 4-post transplant. First, we determined the numbers of donor spleen-derived Thy1.2+ T cells per spleen. The numbers of donor spleen-derived T cells per spleen were significantly lower in Fla recipients compared to PBS-treated recipients [Fla 0.005 ± 0.002 (x103) vs PBS 0.04 ± 0.03(x103), p=0.02]. Accordingly, donor spleen-derived both CD4 and CD8 T cells per spleen of Fla recipients were also found significantly lower compared to PBS-treated recipients (CD4, p=0.04; CD8, p= 0.003). The CD62L, a naïve and also markers for allo-reactive T cells that cause GvHD were found significantly lower in both CD4 and CD8 T cells (CD4, p= 0.03; CD8, p=0.003) and the inducible co-stimulatory molecule 1 (ICOS-1), another prominent T cells activation marker were also found significantly lower (CD4, p= 0.04; CD8, p=0.007) in Fla recipients compared to PBS-treated recipients. These lower immune phenotypes of donor T cells in Fla recipients may reduce the initiation of GvHD at the early time points of transplant. However, flagellin-induced reduction of donor T cells activity were not suppressed considerably as the numbers of donor spleen-derived CD4 T cells expressing activation markers such as CD25 {Fla 0.5 ± 0.2 (x103) vs PBS 4.5 ± 0.5 (x103), p=0.07} and CD69 {Fla 0.5 ± 0.2 (x103) vs PBS 6.5 ± 5.0 (x103), p=0.08} per spleen were not found significantly different. On the other hand, the numbers of CD8 T cells those expressed CD25 {Fla 0.2 ± 0.04 (x103) vs PBS 1.3 ± 0.7 (x103), p=0.01} and CD69 {Fla 0.4 ± 0.1 (x103) vs PBS 2.4 ± 1.7 (x103), p=0.03} per spleen were found significantly lower in Fla recipients compared to PBS-treated recipients. Although over 90% of donor spleen CD4 T cells of both Fla and PBS-treated recipients expressed PD-1, 24% and 32% respectively, expressed IFN-γ after vitro PMA stimulation. Similar results were found in case of CD8 T cells. The over expression of PD-1 in HSCT recipients, thus did not make the donor T cells exhausted as they expanded over 100 times within day 10 post transplant with the expression of PD-1. Although similar level of serum IFN-γ was determined between the Fla and PBS-treated recipients on day 10 post-transplant, IFN-γ level was found below detection limit on day 4 post-transplant. The serum levels of IL-1β and IL-10 were undetectable on both days 4 and 10 post-transplant. Serum LPS were found identical in both Fla and PBS-treated groups determined on day 4 and 10 days post transplant. Conclusion: Flagellin protected allogeneic HSCT recipients from lethal GvHD by reducing donor CD62L+ and ICOS-1+ T cells expansion and activation within 4 days post-transplant without compromising their normal immune responses. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 844 Background: RBC transfusion has clear efficacy in treating various types of anemia. However, in recent decades, there is a renewed focus on the potential for negative clinical sequelae from transfusing stored RBCs. Whether or not older, stored RBC units are associated with adverse outcomes remains controversial. However, there are clear cellular and biochemical data that RBC units accumulate particles and molecules over time with known toxicity when administered to animals and/or humans. Among the most potent of these are prostaglandins and leukotrienes (jointly known as eicosanoids), which are potent mediators of inflammation and vascular pathology. Indeed, arachidonic acid (AA), and 5-, 12-, and 15-hydroxyeicsotetranoic acid (HETE) accumulate during storage of human RBCs and are biologically active in priming neutrophils (Silliman et al., Transfusion 2011 51(12):2549-54). It is well known that there is substantial donor-to-donor variation in how well RBCs store from the standpoint of post-transfusion RBC recovery; however, it is unclear whether the accumulation of eicosanoids varies substantially among donors. If differences exist, then it may be useful to screen RBC units prior to transfusion into patients with illnesses likely to be affected by eicosanoid exposure. Using a well characterized mouse model of RBC storage, and different strains of donor mice, we tested the hypothesis that there are genetic determinants affecting eicosanoid levels in stored RBCs. Methods: RBCs from C57BL/6 (B6) and FVB mice were collected in CPDA-1, filter leukoreduced, and stored under conditions previously shown to model human RBC storage. Samples collected on days 0, 5, 9, and 14 were analyzed by small molecule mass spectrometry. The study was repeated 3 times and combined data were analyzed. Results: AA accumulated over storage time in both B6 and FVB RBC units to a similar level. In contrast, although essentially no accumulation of eicosanoids was observed in B6 RBC units, substantial time-dependent increases (compared to day of collection) were observed in FVB RBC units for 5-HETE (4-fold) and 15-HETE (12-fold). In addition, a greater than 10-fold increase was observed for prostaglandin E2 in FVB RBC units with no detectable prostaglandin E2 in B6 RBC units [ findings were consistent in all 3 experiments and all differences had p values of

Description: Bacground: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Flagellin is a bacterial protein and a TLR5 agonist that showed diverse immunological responses in both human and animal including both activation of dendritic cells and immuno-suppression. We recently observed that prophylactic use of flagellin protected allogeneic HSCT recipient from GvHD without affecting host immune reconstitution. Acute GvHD has been reported to be mediated by allo-reactive CD62L+ T cells, and over 80% of murine naïve splenic CD4+ and CD8+ T cells express CD62L. In order to test the effect of flagellin on GvHD mediated by the CD62L+ CD4+ and CD62L+CD8+ donor T cells, we investigated clinical manifestation of GvHD as well as the in vivo expression of CD62L on donor T cells in flagellin treated versus control treated allogeneic HSCT recipients. Methods: We established a parent →F1 MHC major mismatched model (C57BL/6 → C57BL/6 × BALB/c) for allogeneic HSCT for which GvHD is the major complication. Recipient mice received 5 × 10^6 T cell depleted (TCD) bone marrow cells and 5×10^6 or 10×10^6 CFSE labeled donor splenocytes from naïve C57Bl/6 congenic donors. 50 μg flagellin per recipient was administered intraperitoneally 3 hours before irradiation and 24 hours after allogeneic HSCT (treated). CB6F1 recipients that received no flagellin (untreated) and recipients of syngeneic HSCT were used as control. Recipients were sacrificed on day 66+ transplant and the numbers of CD62L+ T cells and foxp3+CD4+CD25+ T cells were determined by FACS. Recipients of CFSE treated donor splenocytes were sacrificed on day 4 post HSCT, splenocytes were harvested and analyzed for CD62L expression on T cell subsets undergone in vivo cell division by Flow cytometry. 5 mice were used per group. Results: Flagellin treated recipients did not have GvHD and had no mortality. Untreated control recipients had 87% survival at 30 days post transplant and had signs of chronic GvHD. While total cell number and also donor spleen- and BM-derived CD4+ and CD8+ T cells per spleen in untreated recipients were significantly lower compared to flagellin treated recipients (p=0.0006) on day 66 post transplant, persistent of donor spleen-derived CD62L+CD4+ T cells and CD62L+CD8+ T cells per spleen were not significantly different (p=0.13 and p=0.07, respectively). Moreover, higher number of foxp3+CD25+CD4+ regulatory T cells were found in the spleen and thymus in treated recipients compared to untreated recipients. Within day 4 post transplant, the number of CD4+ T cells per spleen of treated and untreated recipients increased significantly compared to syngeneic recipients (p=0.001 and p=0.03, respectively). Although equivalent numbers of CD62L+CD4+ T cells were observed in both treated and untreated recipients (p=0.3), significantly increased numbers of CD62L+CD8+ T cells was found in treated recipients compare to untreated recipients (p=0.02). Moreover, significantly higher numbers of divided (far left CFSE staining population) CD62L+CD4+ and CD62L+CD8+ T cells were found in recipients of treated splenocytes within day 4 post transplant followed by down regulation of CD62L surface marker compared to untreated recipients (p=0.02 and p=0.01, respectively). Conclusion: Flagellin treated recipients had limited GvHD and had rapid increased divided CD4+CD62L+ T cells followed by CD62L-ve activated CD4+ T cells per spleen in treated recipients compared to untreated recipients may be one of the major affect mediated by flagellin. Flagellin-TLR5 receptor agonistic effect may reduce production of biological factor(s) essential to generate allo-reactive T cells or directly stimulate CD62L+CD4+ and CD62L+CD8+ T cells in different activation status other than allo-reactive T cells; maintain a balanced immune reconstitution in lymphoid organs by producing regulatory T cells through their thymus. Therefore, use of flagellin may be a novel therapeutic approach to treat blood cancer patients with allogeneic HSCT without GvHD and toxicity.