Donor Hematopoietic Cell Engraftment Research Articles

Host CD4+CD25+FoxP3+ Regulatory T Cells Facilitate Donor Engraftment Without Impacting Graft Versus Host Disease Onset After Major-Mismatched Hematopoietic Stem Cell Transplantation

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. ResultsCD4+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. ConclusionOur 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.

Expansion of Activated Memory CD4+ T Cells Affects Infectivity of CCR5-Tropic HIV-1 in Humanized NOD/SCID/JAK3null Mice

Humanized mice reconstituted with human hematopoietic cells have been developed as an experimental animal model for human immunodeficiency virus type 1 (HIV-1) infection. Myeloablative irradiation is usually performed to augment the engraftment of donor hematopoietic stem cells (HSCs) in recipient mice; however, some mouse strains are susceptible to irradiation, making longitudinal analysis difficult. We previously attempted to construct humanized NOD/SCID/JAK3null (hNOJ) mice, which were not irradiated prior to human HSC transplantation. We found that, over time, many of the reconstituted CD4+ T cells expanded with an activated effector memory phenotype. Therefore, the present study used hNOJ mice that were irradiated (hNOJ (IR+)) or not (hNOJ (IR−)) prior to human HSC transplantation to examine whether the development and cellularity of the reconstituted CD4+ T cells were influenced by the degree of chimerism, and whether they affected HIV-1 infectivity. Indeed, hNOJ (IR+) mice showed a greater degree of chimerism than hNOJ (IR−) mice. However, the conversion of CD4+ T cells to an activated effector memory phenotype, with a high percentage of cells showing Ki-67 expression, occurred in both hNOJ (IR+) and hNOJ (IR−) mice, probably as a result of lymphopenia-induced homeostatic expansion. Furthermore, when hNOJ (IR+) and hNOJ (IR−) mice, which were selected as naïve- and memory CD4+ T cell subset-rich groups, respectively, were infected with CCR5-tropic HIV-1 in vivo, virus replication (as assessed by the plasma viral load) was delayed; however, the titer subsequently reached a 1-log higher level in memory-rich hNOJ (IR−) mice than in naïve-rich hNOJ (IR+) mice, indicating that virus infectivity in hNOJ mice was affected by the different status of the reconstituted CD4+ T cells. Therefore, the hNOJ mouse model should be used selectively, i.e., according to the specific experimental objectives, to gain an appropriate understanding of HIV-1 infection/pathogenesis.

Exercise promotes bone marrow cell survival and recipient reconstitution post-bone marrow transplantation, which is associated with increased survival

Bone marrow transplantation (BMT) is associated with a high risk of mortality, partially because of the harmful effects of the preconditioning myeloablative regimens. We have recently demonstrated increased bone marrow cell survival and proliferation in response to exercise training, which may be attributable to increased quality of the niche. The purpose of the present study was to determine the extent to which exercise preconditioning of recipients could increase the success of BMT. Recipient mice remained sedentary (SED) or were exercise-trained (EX) on a treadmill (3 d/wk for 8 weeks) before reconstitution with green fluorescent protein (GFP)-labeled donor marrow. Recipient survival, both donor-derived and total (donor- and recipient-derived) blood reconstitution were measured by flow cytometry. The first and fourth day after BMT apoptosis, cellularity and donor cell homing were determined in the recipients' bone marrow cavity by flow cytometry. Whereas only 25% of SED mice survived, 82% of EX recipients survived the BMT. Homing of donor-derived marrow cells to the recipients' marrow cavity acutely after BMT was not altered in EX, but EX mice displayed decreased levels (10%; p < 0.05) of activated caspase-3/-7 one day after BMT, leading to a maintenance of marrow cellularity in mice preconditioned with exercise. The acute inhibition of marrow cell apoptosis in EX mice resulted in increased total blood cell reconstitution at 1 and 3.5 months after BMT in EX mice (42% and 43%, respectively; both p < 0.05). Short- and long-term donor-derived engraftment was not different between EX and SED recipients. Exercise training increases recipient survival after BMT with increased total blood cell reconstitution.

Parathyroid Hormone Stimulates Human Mesenchymal Stem Cells to Enhance the Expansion of Hematopoietic Stem Cells,

Abstract 3407Expanding hematopoietic stems cells (HSCs) has a great interest for successful engraftment of donor cells in hematopoietic stem cell transplantation (HSCT). In addition, mesenchymal stem cells (MSCs) show unique features with multi-differentiation, immune-modulation and tissue regeneration capabilities. Recent studies demonstrated the MSC-derived mature osteoblasts play important roles for the maintenance of HSC niche in the bone marrow (BM) microenvironment. We found that culture-expanded human MSCs support the expansion of HSCs and stimulation of MSCs with parathyroid hormone (PTH) further enhances the MSC-mediated expansion of HSCs.Culture-expanded MSCs (2×104 cells/well) were seeded on a 24-well culture plate. Autologous purified CD34+ HSCs (0.6×103 cells/well) was applied on the MSC-grown plate and co-cultured in StemSpan Serum Free Expansion Medium (StemCell Technologies) supplemented with 100ng/mL SCF, 100ng/mL Flt-3 ligand, 50ng/mL TPO and 20ng/mL IL-3. After 10 days of co-culture of HSCs with MSCs, the number of CD34+ cells was increased to 9.0±1.6×103 (Fig. culture condition #2), whereas that cultured in the absence of MSCs was 0.98–8, −1 and □ {7 of the irradiation. The number of leukocytes was decreased to the bottom level around 6–8 days after the irradiation in both PTH-treated and non-treated (control) mice. However, the number of leukocyte showed a steep increase in PTH-treated mice and it reached maximal level on day 14 at 16±5.2×103/mL compared with that of 6.4±1.4×103/mL in the control mice. In vitro experiments confirmed that PTH has no direct effects on the expansion of HSCs. It is suggested that PTH has supportive effects on hematopoietic recover in vivo, probably in part, through acting on MSCs in BM microenvironment.In conclusion, PTH stimulated MSCs to enhance HSC expansion mainly through contribution of surface membrane molecules. A two-step manipulation of MSCs with culture expansion and subsequent PTH stimulation showed enhancement of MSC-mediated expansion of HSCs. This work revealed the novel mechanisms underlying HSC expansion mediated by MSCs. In addition, this novel concept of MSC manipulation could provide HSCT procedure with new technology for effective engraftment of donor hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

Novel Double Labeled Mixed Lymphocyte in Vitro Assay to Predict the Dominant Graft in 2 Unit UCB Transplantation

Abstract 4699 Introduction:Umbilical cord blood cells (UCB) from allogeneic donors have been established as an alternative source for HSC transplantation in patients who lack suitably HLA matched bone marrow or peripheral blood stem cells from adult donors. Transplantation using 2 unit UCB has been shown to compensate the low engraftment and slow hematopoietic recovery resulting from 1 unit UCB transplantation in full stature adult patients. At present, there are no unit specific factors that reliably predicts for the “winning unit” in 2 unit UCB transplantation, e.g. cell viability, number of infused total nucleated cells, CD34+ or CD3+ cells, sex mismatch, ABO blood group, and degree of HLA mismatch. In vivo mouse models suggest that CD34 negative subsets play an important role. Among CD34 negative subsets, CD8 T subset accounts for approximately 34.0+/−23.3% of T lymphocytes from UCB. In bone marrow transplantation CD8 T cells have been found to facilitate donor hematopoietic cell engraftment. Moreover, it has been reported that 1 dominant unit coincides with a specific CD8 T cell response against the non-engrafted unit which was not observed from CD4 or NK cells. Methods:In this study, we used volunteer donated UCB research units (kindly provided by P. Rubinstein, MD, New York Blood Center). Mononuclear cells (MNC) were purified by Ficoll gradient centrifugation, and CD3 T cells were isolated with CD3 MicroBeads (Miltenyi Biotec; autoMACS). The purified CD3 (confirmed by FACS >95% purity) cells were labeled with CFSE and DDAO-SE. After labeling, the cells from two different donors were mixed in 96-well U-bottom plates for continued culture in 37 °C 5% CO2. The expansion from each labeled donor cells was evaluated using flow cytometry; the dead cells were gated out using propidium iodide, and the data was analyzed using FlowJo software. For proper T cells activation, we also compared different activation conditions using i.) anti-CD3/CD28 Beads, ii.) anti-CD3 antibody plus anti-CD28 antibody, and iii.) cytokine IL-2. The schematic illustration of methods is shown in Figure 1. [Display omitted] Results and discussion:We noted that T cells from UCB are primarily at naïve stage as determined by CD45RA (93.8 +/− 7.11%) and CCR7 (84.9 +/− 12.0%) expression. We also determined the optimal activation condition using a modified mixed lymphocyte reaction from 2 UCB units. Four days after incubation, the proliferation from 2 units labeled with CFSE and DDAO-SE could be reproducibly distinguished using FL1 channel for CFSE and FL4 channel for DDAO-SE (Figure 1). The optimal concentration for labeling using CFSE (1 mM) and DDAO (1 μM or 3 mM) was determined by titration. To avoid cell toxicity resulting from CFSE and DDAO-SE labeling, as well as self-crossing from each donor using two dyes, we examined additional mixed lymphocyte analyses in which each donor was labeled with CFSE or DDAO-SE respectively and vice versa. As shown in Figure 1, we found consistently that the predicated dominant unit accounted for the majority of culture (73.2% stained with DDAO; 63.5% stained with CFSE) after 4 days co-culture. The dominance was not correlated with cell proliferation indicated by the proliferation index (1.12 for dominant and 1.48 for another unit). After confirmation of this in vitro assay, further studies were conducted to evaluate the IFN-γ release of 2 UCB units in this optimized mixed lymphocyte assay in the condition using cytokine IL-2. Interestingly, we could only detect IFN-γ by intracellular staining in one unit when co-culture was set-up using CD3 T cells from each unit; the expression of IFN-γ was not detected when we used CD3 T cells from 1 unit. The correlation between dominance and the expression of IFN-γ is currently under investigation. Conclusion:UCB Transplantation is an important alternative for patients lacking bone marrow or peripheral blood stem cell donors. With the establishment of this novel modified mixed lymphocyte in vitro assay for prediction of the “winning” immune dominant unit, routine analyses can be performed to guide unit selection. Further interventions can be exploited to preferentially treat the expected dominant unit with glycosylation, cytokines, prostaglandins, or C3a compliments to further enhance hematopoietic stem cells trafficking and engraftment to the marrow. Disclosures:No relevant conflicts of interest to declare.

BTLA-HVEM interaction delivers bidirectional signals and modulates the severity of GVHD (169.48)

Abstract B and T lymphocyte attenuator (BTLA), upon interaction with herpesvirus entry mediator (HVEM), delivers an inhibitory co-signal which regulates immune response in various diseases. In graft-versus-host disease (GVHD), unexpectedly, BTLA mediates positive effects on donor T cell survival, but immunological mechanisms of this function have yet to be fully explored. Our recent studies addressed a role of BTLA in GVHD by employing the newly established agonistic anti-BTLA mAb that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. We revealed that BTLA signal inhibited donor anti-host T cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand and delivers HVEM prosurvival co-signal in donor T cells. Thus, BTLA-HVEM interaction delivers positive and negative bidirectional co-signals to donor T cells via HVEM and BTLA, respectively, and modulates the severity of GVHD. Additional data obtained by recent studies will be also presented.

Improved engraftment with minimal graft-versus-host disease after major histocompatibility complex-mismatched cord blood transplantation with photochemically treated donor lymphocytes

There is a significant risk of severe graft-versus-host disease (GVHD) and graft failure after unrelated umbilical cord blood transplantation (CBT) if donor-recipient pairs are mismatched at major histocompatibility complex (MHC) loci. To mitigate these risks after MHC-mismatched CBT, we infused psoralen-treated, photochemically inactivated, mature donor T-lymphocytes with MHC (H2-haplotype) mismatched murine donor fetal near-term peripheral blood (FNPB) cells after sublethal irradiation. We analyzed the rates of donor engraftment, GVHD and long-term survival in H2 haplotype disparate (C57BL/6 [H-2(b)/Thy1.1] → AKR [H-2(k)/Thy1.2]) recipient mice. We observed inconsistent donor engraftment after transplantation with cord blood alone, but superior engraftment and long-term survival after FNPB transplantation supplemented with psoralen-treated donor T-lymphocytes. Additionally, there was fatal GVHD after FNPB co-infusion with untreated donor T-lymphocytes, but minimal GVHD after FNPB supplemented with psoralen-treated donor T-lymphocytes transplantation. Donor MHC(high)/c-Kit(+)/lineage(-)/CD34(-) stem cells were noted in the recipient bone marrow compartment following co-infusion of photochemically inactivated T-cells with FNPB. Despite the non-myeloablative preparation before FNPB infusion, complete hematological recovery was delayed until 50-60 d after transplantation. We observed that co-transplantation of psoralen-treated donor T-lymphocytes with FNPB facilitated durable engraftment of donor hematopoietic stem cells in the marrow and splenic compartments with complete but delayed recovery of all hematopoietic lineages. This CBT model establishes the possibility of ensuring donor engraftment across a MHC barrier without severe GVHD.

Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway

AbstractB and T lymphocyte attenuator (BTLA) is a coinhibitory receptor that interacts with herpesvirus entry mediator (HVEM), and this interaction regulates pathogenesis in various immunologic diseases. In graft-versus-host disease (GVHD), BTLA unexpectedly mediates positive effects on donor T-cell survival, whereas immunologic mechanisms of this function have yet to be explored. In this study, we elucidated a role of BTLA in GVHD by applying the newly established agonistic anti-BTLA monoclonal antibody that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. Our results revealed that provision of BTLA signal inhibited donor antihost T-cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand that delivers HVEM prosurvival signal in donor T cells. Collectively, current study elucidated dichotomous functions of BTLA in GVHD to serve as a costimulatory ligand of HVEM and to transmit inhibitory signal as a receptor.

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.

Caspase Inhibitor ZVAD-fmk Facilitates Engraftment of Donor Hematopoietic Stem Cells in Intra–Bone Marrow–Bone Marrow Transplantation

When bone marrow transplantation (BMT) is carried out, survival of the donor hematopoietic stem cells is crucial to maintain donor hematopoiesis in the recipients. We have shown that intra-bone marrow-bone marrow transplantation (IBM-BMT) can induce the rapid recovery of donor hematopoiesis and allow a reduction in radiation doses as a pretreatment for BMT. If IBM-BMT methodology can be further improved, BMT could be carried out more safely and more easily. In this experiment, we attempted to suppress apoptosis of donor hematopoietic cells using a caspase inhibitor, ZVAD-fmk, upon IBM-BMT in mouse allogeneic IBM-BMT. IBM-BMT with ZVAD-fmk induced superior engraftment of donor hematopoietic cells and greater numbers of day-12 colony-forming units of spleen (CFU-S) than IBM-BMT without ZVAD-fmk upon allogeneic BMT (C57BL/6 into BALB/c mice). ZVAD-fmk slightly suppressed apoptosis of whole BMCs, whereas it significantly suppressed apoptosis of c-kit+/Sca-1+/lineage(-) cells (KSL cells) in vitro. These results suggest that ZVAD-fmk can suppress apoptosis of hematopoietic stem cells and/or immature progenitor cells of the donor bone marrow cells, thereby accelerating the donor hematopoiesis.

Comparison of donor chimerism following myeloablative and nonmyeloablative allogeneic hematopoietic SCT

Surveillance of hematopoietic chimerism following hematopoietic SCT (HSCT) with nonmyeloablative (NMA) preparative regimens is standard to assess the need for clinical intervention. Monitoring of donor chimerism following HSCT with myeloablative (MA) preparative regimens is, however, not considered useful because engraftment is thought to occur rapidly and consistently. This study compares the timing of donor hematopoietic cell engraftment in patients undergoing NMA conditioning with fludarabine and TBI with those receiving MA conditioning with BU- or TBI-based regimens. Achievement of ≥ 90% donor leukocyte chimerism occurred rapidly and consistently in all three groups and time to achievement of ≥ 90% donor T cells was similar among the three groups (P = 0.57). Achievement of ≥ 90% donor leukocyte chimerism was not associated with risk of acute or chronic GVHD, graft rejection, relapse or all cause mortality in multivariate analyses. Donor T-cell chimerism of ≥ 90% was significantly associated with development of extensive chronic GVHD. The value of routine surveillance of chimerism following any of the preparative regimens used in this study should be reevaluated.

Augmentation Of Hematopoietic Engraftment Without Graft Versus Host Disease By “Add-Back” Of Photochemically Treated T Lymphocytes In Mismatched Cord Blood Transplantation

Augmentation Of Hematopoietic Engraftment Without Graft Versus Host Disease By “Add-Back” Of Photochemically Treated T Lymphocytes In Mismatched Cord Blood Transplantation

Improved Engraftment without Graft-Versus-Host Disease After MHC-Mismatched Cord Blood Transplantation with Photochemically Treated Donor Lymphocytes.

Abstract 2425Poster Board II-402 Introduction:Unrelated cord blood transplantation (CBT) is associated with a risk of graft rejection due in part to a limiting cellular content of the CB unit. Increasing the cellular content of the CB unit mitigates the graft rejection risk, but methods to use adjuvant immuno-modulatory cell co-infusions have also been tested with some success. We have investigated the co-infusion of photochemically (psoralen S59) treated mature donor T lymphocytes in a major histocompatibility complex (MHC) [H2-haplotype] mismatched murine transplant model as a new method to facilitate engraftment of donor CB cells. Methods:We analyzed the rates of donor hematopoietic cell engraftment, graft versus host disease (GVHD), and long-term survival in H2 haplotype disparate (C57BL/6®AKR) mice after CBT. Three different experimental groups were transplanted after sublethal radiation. Group 1 received allogeneic full term newborn peripheral blood alone, group 2 was transplanted with the same donor cells and unmanipulated donor T cells, and group 3 was transplanted with the similar donor cells and psoralen (S-59) treated donor T cells. Results:We observed a low rate of donor engraftment after transplantation with cord blood alone (Group 1). There was better engraftment but a high rate of fatal GVHD after transplantation with cord blood and unmodified donor T-cells (Group 2). The best results were observed after transplantation with 3 × 106 nucleated cord blood cells and 9 ×106 S-59 treated T cells (Group 3b). The engraftment rate was 75% compared to 12.5% after transplantation with 6 × 106 CB cells alone (p=0.04). The long-term survival in group 3 was 100% and the rate and severity of GVHD were minimal. Engraftment observed after CBT with unmodified donor T-cells (group 2) was accompanied by severe GVHD and poor survival. Donor myeloid, B cells and T cells were documented in the spleen and bone marrow of Group 3 mice by 30 days after CBT, although full hematological recovery was delayed until 50-60 days after CBT. Conclusions:These results show improved cord blood engraftment kinetics across a disparate H2 haplotype by adding psoralen-treated donor T lymphocytes. Co-transplantation of psoralen treated lymphocytes with CB cells facilitated durable engraftment of donor MHC high/c-kit+ cells in the marrow and splenic compartments with complete but delayed hematopoietic recovery. The low GVHD risk and excellent long-term survival observed in this murine model suggests the potential for clinical application.Treatment GroupCB dose (× 106)T-cell dose (× 106)GVHD (score 1–10)EngraftmentSurvival1 (CB alone)0.5 – 10-1/37 (3)1/37 (2.7%)36/37 (97%)2 (CB + T)1.0 – 3.03.03/8 (6)3/8 (38%)4/8 (50%)3a (CB + S59T)0.5 – 3.01.0 – 3.00/161/16 (6.3%)16/16 (100%)3b (CB + S59T)3.09.01/8 (3)6/8 (75%)*8/8 (100%)3c (CB + S59T)6.03.00/63/6 (50%)6/6 (100%)*p value=0.007, compared to group 1 transplanted with similar nucleated cord blood cell dose. Disclosures:No relevant conflicts of interest to declare.

In Situ Activation and Expansion of Host Tregs: A New Approach to Enhance Donor Chimerism and Stable Engraftment in Major Histocompatibility Complex-Matched Allogeneic Hematopoietic Cell Transplantation

Host antidonor effector T cells represent a major barrier to the successful engraftment of allogeneic donor hematopoietic progenitor and stem cells. Here, administration of a complex of IL-2 and anti-IL 2 antibodies (IAC) significantly enhanced donor chimerism early as well as long-term engraftment following reduced-intensity conditioning (RIC) and allogeneic major histocompatibility complex (MHC)-matched hematopoietic cell transplantion (HCT). Timing of administration of this complex was crucial: administration of IAC post-HCT more efficiently facilitated marrow engraftment than pre-HCT treatment. Donor chimerism persisted to >6 months post-HCT. Importantly, this approach clearly suppressed the emergence of host antidonor CD8 T cells 2 to 3 weeks post-HCT as assessed by tetramer staining. Following in vivo reactivation of IAC-treated and control recipients at >5 months post-HCT with donor antigen, only PBS-treated control marrow allograft recipients responded with tetramer-binding CD8 cells. In total, the present findings support the notion that the transient activation and expansion of host Tregs in situ post-HCT can be explored as a new approach to regulate host alloreactivity posttransplant. Interestingly, direct stimulation of recipient Treg cells in RIC recipients obviated a requirement for exogenous Treg cell transfusion in this model and may represent a viable alternative to, and/or complement the adaptive transfer of Treg populations in clinical HCT.

Outcomes Following Allogeneic Hematopoietic Cell Transplantation (HCT) Using Non-Myeloablative Conditioning with Total Lymphoid Irradiation (TLI) and Anti-Thymocyte Globulin (ATG) Confirm a Low Incidence of Graft Versus Host Disease (GVHD) with Retained Anti-Tumor Activity

We recently translated a murine model of bone marrow transplantation that protected against GVHD yet retained graft anti-tumor reactions to a clinical study of 37 patients using TLI and ATG conditioning with HLA matched related and unrelated donors, and showed a marked reduction in the incidence of acute GVHD (Lowsky et al., NEJM 2005; 353:1321–31). We now report the clinical outcomes following transplantation of a larger set of patients using the same TLI and ATG regimen. One hundred and eleven consecutive patients with hemato-lymphoid malignancies (64 with lymphoid malignancies and 47 with myeloid malignancies) received G-CSF mobilized grafts from HLA matched related (60), unrelated (45) or 1-allele mismatched (6) donors. Of the 64 patients with lymphoid malignancies, 17 (27%) were in complete remission (CR), and 47 (73%) were in partial remission (PR) or were with progressive disease (PD) at the start of the transplant regimen. Of these 64 patients 59 (92%) had advanced stage disease and 32 (50%) had disease relapse after a prior autologous transplant. Of the 47 patients with myeloid malignancies 26 (55%) were in a first CR, 10 (21%) in a second CR and 11 (24%) were beyond second CR or were with residual disease at the start of TLI and ATG conditioning. One hundred and seven (96%) patients achieved multilineage donor hematopoietic cell engraftment within 28 days after HCT, while 4 (4%) patients had primary graft failure. All patients were monitored for manifestations of acute GVHD (aGVHD) with standard scoring scales during the first 100 days after transplantation. The cumulative incidence of clinically significant aGVHD grades II–IV was 2% and 11% for recipients of grafts from related and unrelated donors, respectively. All cases of aGVHD were treated to resolution except in one patient. One hundred and two (92%) patients survived beyond 100 days who were evaluated for the development of chronic GVHD. The cumulative risk for extensive chronic GVHD at three years was 27% with no difference in risk for recipients of related and unrelated grafts. Among the 64 patients with lymphoid malignancies, the follow-up for the first enrolled patient was 2252 days and for the last patient 360 days; 20 of these patients died of which 14 were from relapse of disease. Of the 47 patients in PR or with stable disease at the start of TLI and ATG 30 (64%) converted to a CR of which only 7 patients had any evidence of acute or chronic GVHD. The K-M actuarial EFS according to status of disease at the start of TLI and ATG is shown in figure 1a. For the 47 patients with myeloid malignancies, the period of observation the first and last patient was 2229 and 369 days, respectively; 24 of the 25 patient deaths were due to disease relapse. The K-M actuarial EFS stratified according to status of disease at the start of TLI and ATG is shown in figure 1b. The non-relapse mortality at 1 year for recipients of related and unrelated grafts is 3% and 7%, respectively. These data confirm that TLI and ATG conditioning is well tolerated and associated with low incidences of non-relapse mortality and of acute and chronic GVHD. The high incidence of conversion from PR to CR and the relatively low incidence of disease progression in this group of patients with advanced stage disease suggest the presence of retained graft anti-tumor activity. [Display omitted]

Breaking of CD8+ T Cell Tolerance through In Vivo Ligation of CD40 Results in Inhibition of Chronic Graft-versus-Host Disease and Complete Donor Cell Engraftment

In the DBA/2 --> unirradiated (C57BL/6 x DBA/2)F(1) model of chronic graft-vs-host disease (cGVHD), donor CD4(+) T cells play a critical role in breaking host B cell tolerance, while donor CD8(+) T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8(+) T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8(+) T cell anergy such that subsequently activated donor CD8(+) T cells deleted host CD4(+) T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8(+) T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8(+) T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.

The Detection of Donor-Directed, HLA-Specific Alloantibodies in Recipients of Unrelated Hematopoietic Cell Transplantation Is Predictive of Graft Failure.

Animal studies point to a strong role for MHC-specific antibody as a cause for failed donor hematopoietic cell engraftment, but the role of donor-directed HLA-specific allo-antibodies in human transplants has been controversial. To investigate such a role, we used a retrospective case-control design and studied unrelated donor:recipient pairs whose transplants were facilitated through the NMDP. A total of 37 cases with graft failure and 78 matched control pairs were evaluated. The 37 graft failure cases were selected based on survival beyond 28 days of transplant with no sustained engraftment, and available cryopreserved recipient serum to test for HLA-specific alloantibodies. Up to 3 controls were selected for each case, and matched for disease, disease status, graft type, patient age and year of transplant. Patients had AML, ALL, CML or MDS, 98% received myeloablative conditioning regimens, 100% received T replete grafts, 97% received marrow, and 97% received calcineurin-based GVHD prophylaxis. Patients and donors were retrospectively typed for HLA-A,B,Cw,DRB1,DQB1,DQA1,DPB1,and DPA1 by sequencing or other high resolution typing methods. Stored pre-transplant serum samples (patients and controls) were retrieved from the NMDP Research Repository, and assayed for HLA antibodies by solid-phase FlowPRA (One Lambda, Inc). All positive samples were evaluated for HLA specificity by single-antigen microparticles (LabScreen, One Lambda, Inc). Among the 37 failed transplants, 11 (30%) recipients possessed alloantibodies specific for donor HLA Class I or Class II, compared to only 3 (4%) of 78 controls. HLA-DR or DQ specific antibodies were not detected; hence, all HLA Class II-specific antibodies were directed to DP. Recipients with anti-DP antibodies against the donor mismatched DP were at increased risk of graft failure, indicating the importance of recipient antibodies directed against mismatched donor DP antigens. Exact conditional logistic regression analysis for the presence of either Class I HLA-A,B,Cw or Class II HLA-DP antibodies showed similar findings (Class I alone: OR. 6.31: 95% CI 1.17–62.9; p=0.03, Class II alone: OR 12.00; 95% CI 1.46–551.97; p=0.01, Class I and II combined: OR 19.08; 95% CI 2.72–828.49; p=0.0003). Further analyses were conducted to evaluate a limited set of covariates not accounted for in the case:control study, i.e. patient CMV status, cell dose and HLA-C match. Cell dose and CMV status were independently predictive of engraftment, p=0.01 and 0.03, respectively. No effect was observed for HLA-Cw match (p=0.84). The presence of anti-donor HLA Class I or II antibodies was predictive of engraftment when adjustment was made for either cell dose (OR 15.49; 95% CI 2.06–697.83; p=0.002) or CMV status (OR 7.94; 95% CI 0.97–367.84; p=0.05). In summary, these results indicate that donor-specific HLA Class I or Class II antibodies in recipients of unrelated donor hematopoietic cell transplants are associated with failed engraftment. We recommend that, as a “Standard-of-Practice”, all potential recipients be screened for the presence of HLA class I and class II antibodies including HLA DP. Donors should be excluded if they carry mismatched HLA types against which the patient has specific antibodies.

Long-Term Tolerance to Kidney Allografts in a Preclinical Canine Model

Durable immune tolerance supporting vascularized allotransplantation offers the possibility of extending graft survival and avoiding harmful complications of chronic immunosuppression. Immune tolerance to renal allografts was induced in a preclinical canine model through engraftment of donor hematopoietic cells using a combination of low-dose total body irradiation and a short course of immunosuppression. Subsequently, donor renal allografts were transplanted accompanied by bilateral native nephrectomies. With 5-year follow up, we found normal renal function in all recipients and no histological evidence of acute or chronic rejection. This tolerance does not extend universally to donor skin grafts, however, with two of four animals rejecting delayed donor skin grafts. Hematopoietic chimerism produces durable and robust immune tolerance to kidney allografts, although incomplete tolerance to donor skin grafting.

Accelerated diabetic wound healing in a murine model with the application of multipotent human adipose derived stem cells

RESULTS: SSC expressed low level of MHC I, no CD80, CD86 or MHC II. SSC suppressed allogeneic T cell proliferation, IFNgamma production and inhibited stimulatory function of dendritic cells in MLR. In vivo, SSC and BM cells co-infused at 4 time-points (4xBM SSC) significantly delayed GVHD onset compared to 4xBM (p 0.0016) and enhanced donor hematopoietic cell engraftment. The 4xBM SSC group had significant prolongation in flap survival, compared to the 4xBM (p 0.0007) or to the CsA group (p 0.0006). Histopathologically, flap skin and muscle of 4xBM SSC recipients revealed less mononuclear cell infiltration and normal tissue architecture. Two animals (2xBM SSC and 1xBM SSC groups) recovered from GVHD and flaps were accepted. One rat (2xBM group) showed wound healing (POD 56) after initial rejection.

Haploidentical Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation (HCT) Using Total Lymphoid Irradiation (TLI) and Anti-Thymocyte Globulin (ATG) Conditioning Protects Against Acute Graft Versus Host Disease (GVHD).

Murine models of transplantation established that nonmyeloablative conditioning using repeated low doses of irradiation targeted to lymphoid tissues (TLI) and depletive anti-T cell antibodies protects against GVHD by skewing residual host T cell subsets to favor regulatory natural killer (NK) T cells that suppress GVHD by polarizing donor T cells toward secretion of non-inflammatory cytokines such as IL-4. We recently translated the murine protocol to a clinical study using non-myeloablative TLI and ATG host conditioning with HLA matched related and unrelated donors, and showed a marked reduction in the incidence of acute GVHD while retaining graft anti-tumor activity (Lowsky et al., in Press NEJM). Engrafted donor CD4+ T cells showed a marked increase in IL-4 production as compared to CD4+ T cells from controls. We now adapted the TLI and ATG nonmyeloablative host conditioning regimen to a clinical study of allogeneic HCT using haploidentical matched (3/6 HLA matched) related donors to determine if it will result in donor hematopoietic cell engraftment and also protect against acute GVHD. Blood derived hematopoietic progenitor cells were collected by apheresis from donors mobilized with G-CSF and the product was T cell depleted using CD34+ selection. CD3+ T cells were added back to the donor inoculum according to a dose escalation schedule. The initial T cell dose was 1 x105 CD3+ cell/kg with designated increments based on clinical outcomes of up to a maximum of 1 x107 CD3+ cells/kg. The desired CD34+ cell dose was >5 x 106 CD34+ cells/kg for all patients. Seven patients were transplanted; the median age was 53 years (range 27 to 61 years). Five patients had acute myelogenous leukemia, two with disease in remission and three not in remission at the start of TLI and ATG, one with myelodysplastic syndrome, and one with progressive peripheral T cell lymphoma. The median follow-up for all patients is 265 days with three of seven patients alive and free of disease at the last observation period. Sustained donor hematopoietic cell engraftment was achieved in three of three patients only after the T cell dose was increased to 1 x107 CD3+ cells/kg. No patient developed acute GVHD. None of the three patients receiving the highest dose of T cells had any invasive fungal or viral infections. Monitoring of sorted host T cell subsets before TLI and ATG, and immediately after but before the infusion of donor cells, revealed in five of five patients a highly significant skewing of residual host T cells favoring invariant NK T (CD3+ CD161hi Va24 +Vb11 +) cells. The mean absolute number of host CD3+, and CD4+ and CD8+ T cells decreased by 99, 163 and 121 fold, respectively, immediately after conditioning compared to the absolute numbers before the start of TLI and ATG, whereas the mean absolute number of invariant NK T cells decreased by only 11%. In conclusion, we have determined the conditions for successful hematopoietic cell engraftment using a non-myeloablative regimen of TLI and ATG that appears associated with a reduced aGVHD risk yet retained graft anti-tumor activity. As in the pre-clinical model, we show direct evidence that the low incidence of aGVHD is associated with a significant alteration in residual host T cell subsets markedly favoring invariant NK T cells.