ALBERT

Description: Abstract 1900 GVHD is a major complication of bone marrow transplantation (BMT) and results from donor T cells becoming activated and reacting to host antigens. Recently, lymphocyte activation gene-3 (LAG-3) has emerged as an important molecule, negatively regulating T cell activation and has been proposed to play an important role in CD4+CD25+Foxp3+ regulatory T cell (Treg) function. We investigated the functional in vivo role of LAG-3 in Treg and conventional T cells in murine GVHD with the hypothesis that LAG-3 engagement diminishes alloreactive T cell responses after BMT. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown previously that donor Treg are able to suppress GVHD induced by donor allogeneic conventional T cells (Tcon). The role of LAG-3 in Treg function was evaluated both in vitro and in vivo by directly comparing Treg isolated from LAG-3−/− donor mice to Treg isolated from wild type donors (WT Treg). In vitro, in a mixed lymphocyte reaction assay, LAG-3−/− Treg efficiently suppressed the proliferation of alloreactive T cells in a manner similar to WT Treg. In vivo, a bioluminescent imaging assay (BLI) was utilized that allows for quantitative assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight loss, survival and GVHD score. Both LAG-3−/− Treg and WT Treg were equally potent at suppressing Tcon proliferation as illustrated by BLI of luc+ T cells and demonstrated a significant increase in median survival time (MST) as compared to mice receiving Tcon only (35 days for Tcon vs. 58 and 68 days for WT and LAG-3−/− Treg, respectively, P=0.03), but there was no significant difference in MST between the groups receiving WT and LAG-3−/− Treg. Interestingly, when LAG-3−/− Tcon were used to induce GVHD in the absence of Treg, GVHD lethality was accelerated. Thus, all mice receiving LAG-3−/− Tcon showed decreased survival and significantly lower body weights than mice receiving WT Tcon (P=0.017). GVHD scores of LAG-3−/− Tcon recipients were also significantly higher than WT Tcon recipients at Day 20 post BMT (6.0 vs. 2.2, P=

Description: A major challenge following allogeneic hematopoietic stem cell transplantation (HCT) is to establish persistent engraftment of donor hematopoietic cells. Many strategies have been developed to permit engraftment involving high dose chemotherapy, serotherapy with anti-lymphocyte drugs or myeloablative irradiation resulting in highly toxic conditioning regimens. The introduction of less harmful therapies could result in less toxicity especially in the major mismatched setting and when reduced intensity conditioning is required. While recent studies have explored the mechanisms through which donor-type CD4+CD25+FoxP3+ regulatory T cells (Tregs) restrict the development of graft versus host and host versus graft reactions, less is known about the role of host-type Treg in the transplant setting. In syngeneic and minor mismatched HCT host Tregs comprise a major component of the Treg compartment in the first weeks after transplant. Moreover the transplant of in vitro primed host Tregs can improve donor engraftment in major mismatched models of HCT; therefore host Tregs could be one of the key controllers of the host versus graft reaction mediated by residual host CD4+ and CD8+ conventional T cells (Tcons), possibly influencing graft versus host disease (GvHD) onset and severity. In this study we investigated the role of host Treg after major mismatched HCT to understand their impact in graft facilitation and rejection and in GvHD induction and prevention. We investigated the mechanism through which this cell population works and we explored the feasibility and the effectiveness of host Treg adoptive transfer for cellular therapy in HCT animal models. Results CD4+CD25+FoxP3+ host Tregs persist for at least 28 days after total body irradiation (8 Gy) and transplantation of C57BL/6 (H-2b) T cell depleted bone marrow (TCD BM) into BALB/C (H-2d) mice. Host Treg could be found in spleen, lymph nodes and bone marrow with an increase in the Treg/CD4+ cell ratio. Moreover we observed that these residual host Tregs maintain their suppressive function in vitro if harvested 14 days after transplant and incubated with healthy mouse derived Tcons in a MLR. These results are even more relevant as transplanted mouse derived host Tcons lose their ability to proliferate confirming that host Tregs possess a numeric and functional advantage compared to residual host Tcons. Using FOXP3-DTR mice as hosts we observed that host Treg ablation results in reduced donor chimerism after major mismatched TCD BM transplant (p 〈 0.01, analysis performed 2 months after transplant). At the same time, the absence of host Tregs favors host CD4+ T cell persistence (p 〈 0.001) and delays B cell reconstitution (p 〈 0.001). Furthermore, we hypothesized that host Treg act as an immunological barrier for HSCs, providing a protective immunological niche. Confocal microscopic analysis of femurs performed at day 7 after TCD BM transplant confirmed that hypothesis showing host Tregs clustering in the epiphysis where donor hematopoietic stem cell (HSC) engraftment is mainly detectable. To strengthen these results and to provide a clinical translatable tool, we adoptively transferred 5x105/mouse highly purified unmanipulated host Tregs in a non myeloablative (TBI 5.5 Gy) major mismatched model of rejection. We found that the transferred host Tregs induce persistent full donor chimerism if injected together with a sublethal dose of donor Tcons (5x105/mouse, p=0.016) and transiently enhance donor chimerism in the first three weeks after transplant if injected with low dose interleukin-2 (IL-2, 50,000 IU bid for 7 days; p 〈 0.001) without impacting on GvHD incidence and lethality. The relatively low dose of injected Tregs, the possibility to stimulate and expand them in vivo with IL-2 and the safety of this model provide the first evidence of the feasibility of this clinical approach. Conclusion Our findings indicate that host Tregs facilitate bone marrow engraftment in major mismatched HCT models without impacting GvHD. Notably, our observations on the bone marrow environment after transplant strongly suggest that host Tregs can play a role in building the donor HSC cell niche. Finally host Treg adoptive transfer proved to be feasible and effective in animal models providing a new tool for cellular therapy and clinical translation. Disclosures: No relevant conflicts of interest to declare.

Description: Allografting as a curative approach for many hematological malignancies is hampered by the occurrence of acute graft-versus-host disease (aGvHD). Interleukin (IL)-18 stimulates T helper 1 (Th1) and Th2-mediated immune responses and has been shown to modulate aGvHD. It is still unknown whether increased IL-18 levels during aGvHD are of host or donor origin and how the absence of IL-18 impacts migration and expansion of conventional CD4+CD25− (Tconv) and CD4+CD25+ regulatory (Treg) T cells in vivo. By utilizing IL-18 gene deficient donor versus recipient animals we found that the major cytokine production during the early phase of aGVHD induction was recipient derived, while donor hematopoietic cells contributed significantly less. By generating IL-18−/ − luciferase transgenic mice we were able to investigate the impact of IL-18 on Tconv and Treg expansion and trafficking with in vivo bioluminescence imaging. While migration to secondary lymphoid organs was not significantly impacted by the absence of host IL-18, Tconv but not Treg expansion increased significantly. Absence of host IL-18 production translated into lower IFN-γ levels in the early phase after transplantation. We conclude that host derived IL-18 is a major factor for IFN-γ production that may have a protective effect on CD4+ mediated aGvHD, but is non-essential for Treg expansion in an allogeneic environment.

Description: NKT cells, which are CD1d reactive and express an invariant TCR, are thought to play an immunoregulatory role in suppressing dysfunctional immune reactions including graft vs. host disease (GVHD). Whether non-manipulated donor-type NKT can suppress GVHD following adoptive transfer has not been addressed, nor has the trafficking pattern of NKT in a hematopoetic transplantation (HCT) setting. To determine how effectively NKT proliferate and traffic in a HCT setting, 5.5x105 highly purified (〉95%) NKT (DX5+TCRβ+CD4+) from luciferase positive (luc+) C57BL/6 (H-2b) mice were transferred into lethally irradiated Balb/c (H-2d) recipients along with 5x106 T-cell depleted bone marrow (TCD-BM) from wild-type C57BL/6 mice. Proliferation and migration of luc+NKT was monitored by bioluminescence imaging (BLI). By day 4 after transfer, a prominent signal was observed in the spleen and lymph node (LN) sites. Between days 7 and 10, the NKT migrated to skin, while still remaining present in high numbers in LNs, but decreasing to low levels in spleen. The total photons emitted per mouse reached a peak at approximately 25 days after transplantation, followed by a steady decline. The NKT expansion was more vigorous than that of luc+CD4+CD25+ regulatory T cells (Tregs), which also peak around day 25, but do not show extensive migration to skin. Expansion of NKT was far less than conventional (CD4+ and CD8+) T cells (Tcon), with approximately 10 times more photons/mouse being emitted from 5x105 luc+Tcon as from 5.5x105 NKT. The NKT did not cause GVHD where Tcon rapidly resulted in acute GVHD and animal mortality. To assess the impact of donor-type NKT on GVHD induction by Tcon, we co-transferred various doses of highly purified wt NKT at day 0 with 5x106 TCD-BM, followed by 5x105 luc+Tcon at day 2. Weight and survival of groups were monitored, as well as the proliferation of Tcon by BLI. In groups receiving only Tcon, 50% of the mice died within 2 weeks, and 90% died by day 80. Remarkably, if 2.5x104 sorted NKT were transferred, 100% of the mice survived past day 100. To achieve the same effect with Tregs, 2.5x105 Tregs were required. Mice treated with 2.5x104 NKT lost more weight at early time points than those receiving 2.5x105 Tregs, but both groups recovered from this weight loss and did not exhibit other signs of GVHD (hair loss, hunched back, diarrhea, etc). Interestingly, 2.5x104 NKT caused only a slight reduction in Tcon proliferation, whereas 2.5x105 Treg strongly reduced Tcon proliferation. Surprisingly, when the dose of NKT was increased to 5x104, survival was only 62%, and when increased to 1x105 cells, only 50% of mice survived past day 100. To determine how NKT reduce GVHD, we examined intracellular levels of various cytokines in Tcon with or without 2.5x104 NKT, following HCT. At 8 days after HCT, mice receiving NKT had reductions in the number of IL-17-positive cells (CD4: 2.6% to 0.84%; CD8: 2.5% to 0.66%), and TNFα+ cells (CD4: 45% to 27%; CD8 36% to 24%) in cells from LNs. By day 11, IL-17-positive cells had declined to undetectable levels and TNF levels between groups were equivalent. IFNg levels, which were high in both NKT treated and untreated groups at day 8 (85%–95%), decreased significantly in NKT treated mice by day 11 (CD4: 40%; CD8: 43%), but were still abundant in Tcon only mice (CD4: 78%; CD8: 80%). Together these data indicate that NKT cells persist in vivo upon adoptive transfer and suppress GVHD by decreasing the percentage of Tcon secreting pro-inflammatory cytokines.

Description: Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) during allogeneic bone marrow transplantation (BMT). Previous studies in allogeneic BMT models have shown NK cell trafficking to and proliferation in lymphoid organs and GVHD target organs, which are also sites of donor T cell trafficking. This study aims to investigate the impact of NK cells on alloreactive, GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H–2b) or FVB (H–2q) animals were transplanted along with T cell-depleted bone marrow into lethally irradiated BALB/c (H–2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells from the same donor strain (NK+Tcon group). Control mice received lethal irradiation and T cell-depleted bone marrow on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of NK+Tcon mice revealed a significantly lower T cell bioluminescent signal (p=0.03 for FVB into BALB/c on day 6) than from Tcon mice. CFSE proliferation analysis of alloreactive T cells on day 3 after transplant showed no significant change in the percent of donor T cells that have divided in the spleen, and only a slight decrease in the percent of T cells that have divided in the lymph nodes when NK cells are present. However, at this timepoint 82% of the proliferating cells have divided past the third generation, in contrast to 64% in the NK+Tcon mice. Donor T cells in both groups become equally activated in vivo, expressing similar levels of the early-activation marker CD69. T cells re-isolated from NK+Tcon animals at day 5 stained 2 to 10-fold higher for the TUNEL apoptosis marker than those from Tcon mice in the mesenteric and peripheral lymph nodes, respectively (p

Description: Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) following murine allogeneic bone marrow transplantation (BMT). Prior studies have shown that NK cells suppress GVHD by eliminating recipient dendritic cells. To assess additional potential mechanisms of GVHD suppression we evaluated the impact of donor NK cells on GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H-2b) or FVB (H-2q) animals were transplanted along with T cell-depleted bone marrow (TCD-BM) into lethally irradiated BALB/c (H-2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells (Tcon) from the same donor strain (Tcon+NK group). Control mice received TCD-BM on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of Tcon+NK mice revealed a significantly lower T cell bioluminescent signal compared to Tcon mice (p=0.01 on day 5 post T cell transplant). We assessed the impact of NK cells on donor T cell activation and proliferation. CFSE proliferation analysis of alloreactive CD4 and CD8 T cells reisolated on day 4 post transplant showed a decreased percentage of dividing donor T cells in the Tcon+NK group. A reduced percentage of T cells in the Tcon+NK group as compared to the Tcon group expressed the T cell activation marker CD25 (11% and 49%, respectively, among donor CD4) and a reduced percentage of T cells from the Tcon+NK group down-regulated CD62L. Reisolated donor T cell numbers were reduced in the Tcon+NK mice compared to Tcon control mice. The impact of donor NK cells on donor Tcon function was addressed by intracellular cytokine staining. Fewer donor T cells reisolated from the spleen and lymph nodes of Tcon+NK mice produced the proinflammatory cytokines IFN-γ and IL-2 on day 3 after transplant. These observations can be explained by an NK cell-mediated induction of apoptosis in the donor Tcon. T cells reisolated from the peripheral lymph nodes of Tcon+NK animals at day 4 post transplant stained higher for the TUNEL apoptosis marker than those from Tcon mice (p

Description: Abstract 1334 Poster Board I-356 Previous work has demonstrated that both rapamycin (RAPA) and IL-2 enhance CD4+CD25+Foxp3+ regulatory T cells (Treg) proliferation and function. We investigated whether the combination of RAPA and IL-2 administered in vivo could reduce acute graft-versus-host disease (aGVHD) induction and increase survival after bone marrow transplantation (BMT) by induction of Treg. T cell depleted bone marrow (TCD-BM) cells from wild type C57BL/6 mice were injected after lethal irradiation with (800 cGy) into Balb/c recipients on day 0. To induce aGVHD and evaluate the proliferation of donor T cells by in vivo bioluminescence imaging, 1 × 106 conventional CD4+ and CD8+ T cells (Tcon) from luciferase-expressing (luc+) transgenic C57BL/6 were injected intravenously on the same day. RAPA was administered intraperitoneally (dose of 0.5 mg/kg/day) for 14 days and IL-2 (dose of 5 × 104 IU) for 3 days (twice a day). RAPA plus IL-2 significantly reduced the expansion of luc+ Tcon more than RAPA (P=0.04) or IL-2 alone (P=0.002) or no treatment (P = 0.01). Weight loss and aGVHD score were significantly reduced in the mice which were injected with the combination of RAPA and IL-2 compared with those animals injected with RAPA (P = 0.03) or IL-2 alone (P = 0.05). The percentage of donor type CD4+CD25+ cells was increased and CD4+CD25− T cells were reduced from thymic and extrathymic tissues after treatment with RAPA plus IL-2 on day 7 after BMT. The combination of RAPA and IL-2 resulted in a 2.4 fold (mesenteric LN), 2.7 fold (peripheral LN), 4.2 fold (spleen) and 2.1 fold (thymus) increase in the percentage of donor type CD4+CD25+Foxp3+ Treg. In animals receiving the combination of RAPA and IL-2, the percentage of CD4+CD25+Foxp3+ T cells increased from 8.98% (RAPA alone) to 24.2% (RAPA plus IL-2) at 7 days after BMT, representing a 2.7 fold expansion. The cell numbers of CD4+ and CD8+ T cells and CD4+CD25− T cells were reduced and CD4+CD25+Foxp3+ Treg were expanded in the thymus and extrathymic tissues after administration RAPA and IL-2. To study the origin of the expanded Treg in RAPA plus IL-2, BALB/c recipient mice were injected with purified donor CD4+CD25highFoxP3+ Treg and CD4+CD25− and CD8+CD25− (both Foxp3−) Tcon after lethal irradiation followed by RAPA plus IL-2 for 7 days. The combination of RAPA and IL-2 increased the expansion of donor type CD4+CD25+Foxp3+ Treg, but did not result in an increase in the conversion of Foxp3+ Treg from donor CD25− Tcon. We also evaluated whether Foxp3+ non-Treg were present in the expansion of Treg by RAPA plus IL-2 by evaluating interferon-γ- and IL-2-production which was minimally detected in the Foxp3+ cells. In conclusion, the combination of RAPA and IL-2 after BMT synergistically promoted the expansion of donor CD4+CD25+Foxp3+ Treg resulting in prevention of lethal aGVHD. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3750 Graft-vs-Host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation (HCT). Murine models have been critically important to define the biological mechanisms and potential pathways of intervention of GVHD prevention and treatment. Although it is well recognized that GVHD occurs in response to minor histocompatibility antigens, little is know about the kinetics of donor T cell proliferation and homing in minor mismatch models. This is in contrast to models across major histocompatibility barriers where the early development of GVHD has been more thoroughly characterized. In prior studies across major barriers, we have defined an initiation phase within the first 3 days where conventional CD4+ and CD8+ T cells (Tcon) home to secondary lymphoid tissues, proliferate and up-regulate key homing markers allowing for entry into GVHD target tissues during the effector phase (Beilhack, et al. Blood 106:1113, 2005). Since minor models are more similar to clinical HCT, it is critical to understand the timecourse of GVHD development across minor histocompatibility barriers. Since the manifestations of GVHD in recipients of minor mismatch transplants are delayed, it is possible that disease development has altered kinetics. To investigate the temporal and spatial events of donor T cell activation and homing, side-by-side transplants were conducted using T cell depleted bone marrow (TCD BM) and Tcon from donor C57BL/6 (H2b) mice into either major mismatched Balb.c (H2d), or minor mismatch Balb.b (H2b) recipients. Balb.c mice received 1×106 Tcon while Balb.b mice were given 15×106 Tcon, based on previous titration experiments. Recipient mice were regularly scored for GVHD symptoms and monitored for at least 100 days for survival. Additionally, donor Tcon proliferation and migration were monitored longitudinally using in vivo and ex vivo bioluminescent imaging (BLI) by quantitating photons emitted by luciferase (luc+) expressing donor Tcon isolated from luc+ transgenic mice. Donor Tcon were also labeled with CFSE to determine proliferation kinetics at selected timepoints. The upregulation of T cell activation and tissue specific homing markers was examined using flow cytometric analysis of donor CD4+ and CD8+ T cells re-isolated from the secondary lymphoid tissues of transplanted mice. In both models, T cells initially home to secondary nodal sites by 3 days post-transplant, with an exodus into the tissues by day 6, albeit to a lesser extent in recipients of minor mismatch transplants. Additionally, similar levels of global donor CD4+ and CD8+ T cell proliferation between the models were observed using both BLI and CFSE staining as early as 3 days after transplant (BLI, p〉0.05, n=9). More noticeable reductions in minor mismatch recipients were apparent by day 6 (BLI p

Description: Graft-versus-host disease (GVHD) occurs when transplanted donors' T cells recognized the recipients' antigens and damaged host tissues and cells, particularly the skin, gut and liver in the acute setting. Although it is well known GVHD is more aggressive and manifests more quickly across major versus minor histocompatibility barrier, little is known comparatively about the donor T cell activation and T cell repertoire changes. To investigate temporal and spatial events of GHVD development, side-by-side transplants were conducted into major and minor-mismatched murine recipients (Balb.c and Balb.b) using hematopoietic cells from the same donor strain(B6). In both models, T cells home to nodal sites by day 3, proliferate, and exit to GVHD target tissues by day 6. Additionally, expression of homing and activation markers was equivalent for all markers examined on day 3. However, tissue migration and proliferation were reduced in the minor model. By day 6, minor-mismatched T cells had increased CD62L retention and reduced P-selectin and CD44 expression. We also found fewer MHC-matched T cells producing IFN-g and TNF-a. Our data show that early events of donor T cell activation are similar in both models, suggesting that the delayed onset and attenuated disease GVHD seen across minor barriers arise from temporal differences in the effector phase, rather than the initiation phase, of GVHD. To further understand the differences across major versus minor histocompatibility barriers on the T cell repertoire and patterns of T cell alloreactivity, we collected a sample of T cells from donor mice used for transplantation, and also sampled gut tissues from syngeneic, major and minor- mismatched transplanted mice on day 9, 9 and 30 respectively at times when the allogeneic groups of mice showed severe GVHD symptoms. To reduce the background of high percentage of TCR pseudogenes in mouse genome, 5'RACE starting from RNA samples and deep sequencing of TCRa and TCRb were applied to investigate whether TCR repertoire of the major and minor-mismatched mice were skewed with clonal expansion, and how among the major and minor-mismatched mice. While we hypothesized that the major MHC mismatched group would have lower diversity because of expanded clones associated with the GVHD, the shannon index of TCRa indicated gut TCR repertoire of major and minor mismatched mice have greater diversity than syngeneic group(P

Description: 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. Disclosures No relevant conflicts of interest to declare.