Murine Hematopoietic Stem Cell Transplantation Research Articles

Posttransplant cyclophosphamide contributes to the impairment of the graft-versus-leukemia effect and the amelioration of graft-versus-host disease with the suppression of alloreactive T cells in a murine stem cell transplant model

Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for graft-versus-host disease (GVHD) due to its suppression of donor-derived alloreactive T cells in allogeneic hematopoietic stem cell transplantation (HSCT). The graft-versus-leukemia (GVL) effect is an antileukemia effect induced by donor-derived alloreactive T cells, similar to GVHD, whereas no studies have demonstrated the association between the dynamics of donor-derived alloreactive T cells and impairment of the GVL effect after HSCT with PTCy. We herein evaluated the dynamics of donor-derived T cells expressing a functional marker for alloreactivity, programmed cell death-1 (PD-1), in a murine HSCT model with PTCy. We showed that PTCy was associated with the development of leukemia cells and the decreased survival probability in an HSCT model with leukemia cells, whereas PTCy could ameliorate GVHD and increased the survival probability in the HSCT model without leukemia cells. We revealed that the percentages of PD-1 expressing donor-derived CD8+/CD4+ alloreactive T cells, with the exception of CD44+ memory T cells, in the recipient spleen were suppressed with PTCy, and that donor T-cell chimerism levels were decreased early after HSCT with PTCy. Our results suggest that PTCy correlated with the impairment of the GVL effect and the amelioration of GVHD through the suppression of PD-1 expressing donor-derived CD8+/CD4+ alloreactive T cells after HSCT.

CD45-ADC Plus Janus Kinase (JAK) Inhibitors As Conditioning for MHC-Mismatched Murine Hematopoietic Stem Cell Transplantation Is Associated with Minimal Toxicity and Graft Versus Host Disease

INTRODUCTION: Allogeneic hematopoietic stem cell transplantation (alloHSCT) provides the best chance for disease control in acute myeloid leukemia (AML). The known anti-leukemia benefit of alloHSCT is attributable to pre-transplant conditioning with chemotherapy and/or radiation and the graft-versus-leukemia (GvL) effect. However, the utility of alloHSCT is limited by significant treatment-related morbidity and mortality resulting from conditioning regimen-related toxicities and graft versus host disease (GvHD). AML is primarily diagnosed in elderly patients, for whom alloHSCT may be contraindicated due to an inability to tolerate these adverse effects. Therefore, an unmet clinical need exists for novel alloHSCT conditioning regimens that minimize toxicity without sacrificing therapeutic efficacy. Previously, we and others (Palchaudhuri et al. (2016), Nat Biotech; Persaud et al. (2018), Blood) have shown that antibody-drug conjugates (ADCs) composed of a CD45.2-specific antibody linked to the ribosome-inactivating protein saporin (CD45-SAP) permitted stable, high-level engraftment in murine syngeneic HSCT. We hypothesize that such targeted, immunotherapeutic strategies can be applied to the setting of alloHSCT, enhancing its safety and applicability to the treatment of hematologic diseases. METHODS: For engraftment studies, recipient mice were conditioned with 3 mg/kg CD45-SAP 7 days prior to infusion of 10-20 x 106 whole bone marrow cells. The following alloHSCT models were used: MHC-matched (BALB/c→DBA/2), F1-to-parent (CB6F1→B6), and F1-to-F1 (B6CBAF1→CB6F1). In some experiments, CD45-SAP treatment was combined with CD4+ and CD8+ T cell depletion and/or the Janus kinase (JAK) inhibitor, baricitinib. For GvHD studies, a parent-to-F1 (B6→CB6F1) model was used in which recipients were infused with 25 x 106 donor splenocytes after CD45-SAP treatment or sublethal irradiation. For studies with human CD45-SAP, MOLM13 AML cell line viability was measured with XTT assays, and cord-blood CD34+cell colony formation was assessed using methylcellulose assays. RESULTS: CD45-SAP combined with CD4+ and CD8+ T cell depletion allowed high-level engraftment in MHC-matched and F1-to-parent alloHSCT models (Figure 1A-B). Baricitinib, previously shown by our lab to prevent GvHD while preserving the GvL effect (Choi et al. (2018), Leukemia), permitted short-term HSC engraftment in F1-to-parent alloHSCT when mice were dosed daily with the drug during the first 21 days post-HSCT (Figure 1C). Graft stability in baricitinib-treated mice was improved by either combining daily baricitinib treatment with pre-transplant T cell depletion (Figure 1D), or by delivering baricitinib via continuous infusion (Figure 1E). In parent-to-F1 GvHD models, CD45-SAP conditioned mice showed minimal morbidity or mortality when infused with allogeneic splenocytes, whereas mice conditioned with sublethal irradiation succumbed within 3 weeks to severe GvHD (Figure 1F). That alloreactive T cells did not cause GvHD in CD45-SAP treated mice prompted us to use donor lymphocyte infusions to enhance engraftment in a fully haploidentical (F1-to-F1) HSCT model. Our results suggest that addition of donor T cells to the bone marrow graft promotes donor engraftment without eliciting clinically apparent GvHD (Figure 1G). Finally, anti-human CD45 (clone BC8), when conjugated to saporin, inhibited MOLM13 cell growth and cord blood CD34+ cell colony formation in vitro (Figure 1H-I), consistent with both an anti-stem cell and anti-leukemic effect. CONCLUSIONS: Combining CD45-SAP with T cell depletion and/or pharmacologic immunomodulation with the JAK inhibitor baricitinib effectively conditions mice for engraftment of MHC-matched and haploidentical HSCs without causing significant toxicity. Strikingly, mice conditioned with CD45-SAP, unlike mice conditioned with irradiation, did not develop GvHD when infused with allogeneic splenocytes. Finally, we have demonstrated that human CD45-SAP is cytotoxic to both the MOLM13 AML cell line and cord-blood derived stem cells. Taken together, these studies provide key proof-of-principle evidence that targeted immunotherapeutics (CD45-SAP plus JAK inhibitors) can accomplish the goals of alloHSCT - creating marrow space for donor HSCs, overcoming immune barriers to engraftment, and eliminating leukemia cells - with limited toxicity or GvHD. Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. DiPersio:Incyte: Consultancy, Research Funding; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Magenta Therapeutics: Equity Ownership; Karyopharm Therapeutics: Consultancy; Bioline Rx: Research Funding, Speakers Bureau.

3118 - GPRASP Family Members Function as Novel Negative Regulators of Murine Hematopoietic Stem Cell Transplantation via Controlling CXCR4 Degradation

Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for many patients with leukemia and hematologic disease. Sadly, immunological complications and infections contribute to high morbidity and mortality post-transplant. Recent work suggests that transplanted HSC are subject to distinct molecular regulation compared with HSC at steady-state. Thus, we seek to identify novel molecular regulators of HSCT. Here we report that multiple GPRASP (G-protein coupled Receptor (GPCR) associated Sorting Proteins) family members function as novel barriers to HSCT in mice. Treatment of hematopoietic stem-progenitor cells (HSPCs) with Gprasp1 or Gprasp2-shRNAs results in up to a 4-fold increase in hematopoietic reconstitution by 4 weeks post-transplant and maintained for more than 16 weeks post-transplant. GPRASPs regulate GPCR degradation by modulating endosomal/lysosomal sorting. The GPCR, CXCR4, a master regulator of HSC engraftment, cell cycle and survival, contains a C-terminal GPRASP-binding motif, implicating it as a target. Gprasp1- and Gprasp2-shRNAs increased survival and quiescence in HSPCs ex vivo and 20 days post-transplant. In contrast, Gprasp loss in Cxcr4-/- HSPCs had no effect on their survival or cell cycle status. Further, Gprasp1 and 2 losses increased CXCR4 levels in HSPCs and perturbed CXCR4 cellular localization. In pilot studies, GPRASP2 appears to physically interact with CXCR4. These data suggest that loss of GPRASP1 or GPRASP2 in HSPCs stabilizes CXCR4, resulting in increased engraftment, survival and quiescence acutely post-transplant. In sum, we have discovered that multiple members of the GPRASP family function as barriers to optimal HSCT by effecting HSC survival, cell cycle status and engraftment via regulation of CXCR4 stability. This protein family has never before been implicated in hematopoiesis or HSC biology. Further, our data illuminate a novel means to modulate CXCR4 levels and open up new avenues to developing therapies that improve HSCT.

T cells take directions from supporting cast in graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only treatment option for several severe hematological malignancies. The development of graft-versus-host disease (GVHD) is a common complication of the procedure and results when donor T cells become activated against recipient-specific antigens. The factors that drive the alloreactive T cell response are not completely understood. In this issue of the JCI, Chung and colleagues present evidence that stromal cells within lymphoid tissue express the Notch ligands Delta-like 1/4 (DLL1 and DLL4), which in turn directly activate T cells. Importantly, inhibition of DLL1/DLL4-mediated Notch signaling in murine HSCT models dramatically reduced GVHD and improved graft survival.

Targeting the Canonical Nuclear Factor-κB Pathway with a High-Potency IKK2 Inhibitor Improves Outcomes in a Mouse Model of Idiopathic Pneumonia Syndrome

Idiopathic pneumonia syndrome (IPS) is a noninfectious inflammatory disorder of the lungs that occurs most often after fully myeloablative allogeneic hematopoietic stem cell transplantation (HSCT). IPS can be severe and is associated with high 1-year mortality rates despite existing therapies. The canonical nuclear factor-(NF) κB signaling pathway has previously been linked to several inflammatory disorders of the lung, including asthma and lung allograft rejection. It has never been specifically targeted as a novel IPS treatment approach, however. Here, we report that the IκB kinase 2 (IKK2) antagonist BAY 65-5811 or “compound A,” a highly potent and specific inhibitor of the NF-κB pathway, was able to improve median survival times and recipient oxygenation in a well-described mouse model of IPS. Compound A impaired the production of the proinflammatory chemokines CCL2 and CCL5 within the host lung after transplantation. This resulted in significantly lower numbers of donor lung infiltrating CD4+ and CD8+ T cells and reduced pulmonary inflammatory cytokine production after allograft. Compound A's beneficial effects appeared to be specific for limiting pulmonary injury, as the drug was unable to improve outcomes in a B6 into B6D2 haplotype-matched murine HSCT model in which recipient mice succumb to lethal acute graft-versus-host disease of the gastrointestinal tract. Collectively, our data suggest that the targeting of the canonical NF-κB pathway with a small molecule IKK2 antagonist may represent an effective and novel therapy for the specific management of acute lung injury that can occur after allogeneic HSCT.

Dysfunction of bone marrow vascular niche in acute graft-versus-host disease after MHC-haploidentical bone marrow transplantation.

Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4+, but not CD8+ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4+ donor T cells through the Fas/FasL pathway.

Both Host-Type and Donor-Type Mast Cells Protect Against Acute Graft-Versus-Host Disease

Originally mast cells have been known as effector cells in the IgE-mediated allergic responses. In addition, recent studies demonstrated that mast cells exerted pro-inflammatory or anti-inflammatory effects in complicated immune response depending on the situation. In allogeneic hematopoietic stem cell transplantation (HSCT), exact role of mast cells in graft-versus-host disease (GVHD) remains unclear. First we investigated whether host mast cells protect acute GVHD or not in a murine HSCT model using mast cell deficient mice. When lethally irradiated wild type (WT) B6 or mast cell deficient mice were transplanted with bone marrow cells and spleen cells from WT Balb/c mice, mast cell deficient B6 host mice showed significantly lower survival rate than WT B6 mice (p<0.01) due to acute GVHD. Moreover when irradiated mast cell deficient host mice (B6 derived) were given BM cells and splenocytes from mast cell deficient Balb/c mice, these mice died much faster compared to the mice receiving WT BM plus splenocytes (p<0.01) as shown below. Next, we demonstrated that bone marrow derived cultured mast cells (BMCMCs) inhibited mixed lymphocyte reaction (MLR) in a dose-dependent manner, when added to the coculture (Stimulator: dendritic cells (DCs) from B6, Responder: spleen cells of Balb/c, BMCMCs from B6). In addition when mast cells generated from the same strain of responder cells (Stimulator: DCs from B6, Responder: spleen cells from Balb/c, BMCMCs from Balb/c) were added to the MLR mixture, MLR was also suppressed even in this condition. Taken together, we have clearly demonstrated that both host-type and donor-type mast cells suppressed alloreaction in vivo and in vitro. However when we used anti-CD3 and anti-CD28 monoclonal antibodies to stimulate T-cells instead of DCs, BMCMCs could not suppress the lymphoproliferation any more. It suggested that mast cells could suppress MLR via the regulation of DCs directly, not T-cells. Next we have shown that BMCMCs dominantly regulated the alloreaction in a cell contact-dependent manner by using transwell system, though mast cells contain many kinds of substances to regulate immune reactions such as IL-10. DCs highly expressed costimulatory molecules such as CD80, CD86, CD40, I-A, however expression level of these molecules was not changed during coculture of BMCMCs and DCs. Finally, we compared the MLR suppression rate between naïve and activated BMCMCs using IgE and specific antigens, however no difference was observed between them. In conclusion, both host and donor-type mast cells have a protective role against acute GVHD.▪ Disclosures:No relevant conflicts of interest to declare.

Signal One and Two Blockade Are Both Critical for Non-Myeloablative Murine HSCT across a Major Histocompatibility Complex Barrier

Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

Inhibition of c-Rel Signaling: A Novel Small Molecule-Based Therapy Diminishing T Cell Alloactivation While Preserving Anti-Tumor Activity

Abstract 454Allogeneic hematopoietic stem cell transplantation (HSCT) was initially developed as a rescue from therapy-related bone marrow failure following high dose chemo/radiation therapy to rescue from therapy-related bone marrow failure, the emphasis has shifted towards allogeneic HSCT as a strategy to facilitate graft-versus-tumor (GVT) activity. Strategies to suppress graft-versus-host diesase (GVHD) are often associated with broader suppression of the immune system leading to immune deficiency and compromised GVT activity.The aim of this study was to modulate T cell responses in the context of GVHD by targeting the NF-kappaB family member c-Rel, a transcription factor that upon antigen receptor triggering regulates lymphocyte survival and proliferation. In T cells, the main target gene of c-Rel is interleukin 2 (IL-2), a cytokine required for normal T cell proliferative and differentiative responses. The effect of c-Rel deficiency on T cell differentiation and function has been investigated previously demonstrating that c-Rel deficient T cells show reduced Th1, Treg and Th17 but normal Th2 responses. Inhibition of c-Rel is therefore expected to impair T cell activation, differentiation, and function. Using methods to inhibit the c-Rel pathway we developed a novel strategy to minimize the severity of GVHD while leaving GVT activity intact.We investigated the role of c-Rel during GVHD in MHC mismatched as well as MHC matched, minor antigen mismatched (more clinically relevant) murine HSCT models. c-Rel−/− T cells caused significantly less GVHD than normal T cells, as determined by survival (p<0.001), clinical GVHD score (p<0.01), weight loss, and histopathology of GVHD target organs (p<0.001 liver; p<0.001 gut, p<0.05 skin). Time course analyses revealed that 3 days post transplant, proliferation and activation of c-Rel−/− T cells was impaired (p<0.001 CFSElow/high ratio; p<0.01 CD25+ donor cells) and pSTAT5 expression was decreased (p<0.05), resulting in less expansion of donor derived effector T cells by day 7 after transplant (p<0.001). Interestingly, serum levels of IL-2 were increased on day 7, likely secondary to decreased pSTAT5-mediated negative feed back on IL-2 secretion, and we observed increased numbers of donor derived Tregs with increased CD25 expression in recipients of c-Rel−/− T cells (p<0.05), suggesting that the increased IL-2 levels primarily benefited the expansion of Tregs. Levels of Th2 cytokines (IL-4, IL-5 and IL-13) were increased and GATA-3 expression was up-regulated in recipients of c-Rel−/− T cells (p<0.05), implicating a shift toward Th2 responses.We next evaluated if c-Rel−/− T cells were able to mediate antitumor activity. We challenged allogeneic HSCT recipients with either a liquid tumor (A20 lymphoma) or a solid tumor (RENCA-renal cell carcinoma) and found that GVT activity in c-Rel−/− T cells recipients was intact in the absence of GVHD, resulting in significantly improved survival compared to recipients of wildtype T cells (p<0.001 A20; p<0.01 RENCA). Using a syngeneic GVT model with EL4 (T cell lymphoma) we could demonstrate strong anti-tumor activity of c-Rel−/− T cells in the absence of T cell alloactivation, reinforcing the notion of separation of GVHD from GVT activity through inhibition of c-Rel signaling.The potential for clinical translation of our approach is highlighted by a series of experiments testing the efficacy of a recently developed Pyrimidinetrione-based small molecule c-Rel inhibitor compound. We found that incubation of T cells with this compound resulted in efficient downregulation of c-Rel expression as well as IL-2 expression in vitro. When we administered T cells that were pre-incubated with this c-Rel antagonizing small molecule, we were able to reproduce our above described findings regarding the effect of c-Rel deficient T cells on GVHD (Figure 1). Importantly, normal T cells pre-incubated with a c-Rel antagonist mediated intact GVT activity, indicating that they are viable and functional T cells. This strongly suggests that the diminished capacity of these cells to induce GVHD is due to the c-Rel inhibitory mechanism of the small molecule compound and not due to a non-specific cytotoxic effect of the in vitro manipulation procedure.Taken together, our findings identify c-Rel as a promising target for the development of a clinical strategy to prevent or ameliorate GVHD while preserving GVT activity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Allogeneic bone marrow grafts with high levels of CD4+CD25+FoxP3+ T cells can lead to engraftment failure

Regulatory CD4(+) CD25(+) FoxP3(+) T cells (T(regs) ) suppress immunological reactions. However, the effect of adding T(regs) to hematopoietic stem cell grafts on recovery and graft versus host disease (GvHD) is unknown. T(regs) from splenocytes of C57Bl/6 and Balb/c wild-type mice were isolated by MACS separation and analyzed by flow cytometry. Using a murine syngeneic transplantation model that clearly distinguishes between donor and host hematopoiesis, we showed that co-transplantation of bone marrow cells (BMCs) with high levels of T(regs) leads to a 100% survival of the mice and accelerates the hematopoietic recovery significantly (full donor chimerism). In allogeneic transplantation, bone marrow and T(regs) co-transplantation were compared to allogeneic bone marrow transplantation with or without the addition of splenocytes. Survival, leukocyte recovery, chimerism at days -2, 19, 33, and 61 for murine CD4, human CD4, HLA-DR3, murine CD3, murine CD8, murine Balb/c-H2K(d) , murine C57Bl/6-H2K(b) , and GvHD appearance were analyzed. Allogeneic bone marrow transplantation requires the addition of splenocytes to reach engraftment. Mice receiving grafts with bone marrow, splenocytes and high levels of allogeneic T(regs) died within 28 days (hematopoietic failure). Here, we show also detailed flow cytometric data reagarding analysis of chimerism after transplantation in unique murine hematopoietic stem cell transplantation models. Our findings showed that the syngeneic co-transplantation of CD4(+) , CD25(+) , FoxP3(+) T-cells and BMCs induced a stimulating effect on reconstitution of hematopoiesis after irradiation. However, in the allogeneic setting the co-transplantation of T(regs) aggravates the engraftment of transplanted cells.

Development of Antigen Specific T Cells After HLA-Mismatched Umbilical Cord Blood Transplantation

Abstract 4544 [Introduction]Hematopoietic cell transplantation (HCT) is a promising strategy for the treatment of both hematologic malignancies and non-malignant hematologic dyscrasias. However, HCT is sometimes complicated by infectious diseases, especially those caused by virus. Although the most important defense mechanism to control viral replication is inducing virus-specific cytotoxic T-lymphocytes (CTLs), the details of CTL development are not fully elucidated in human transplantation. In 1978, Zinkernagel, et al., clarified in murine hematopoietic stem cell transplantation models that MHC-restricted T cells are not governed by donor HLA expressed in bone marrow stem and progenitor cells. Instead, MHC restriction is acquired by interaction with recipient thymic epithelial cells. For this reason, virus-specific T cells are not induced by the mismatched HLA. Due to the increasing number of cord blood and haplo-identical transplantations, many transplantations are being performed under HLA-mismatched conditions. However, the full repercussions of this HLA-mismatch on viral immunity are still unknown. [Clinical case / Methods]For 4 years we followed a 35-year-old patient (HLA-A*24:02, A*33:03) with acute myeloid leukaemia of subtype t(8;21)(q22;q22);RUNX1(AML1)-RUNX1T1(ETO) of the 2008 WHO classification who underwent cord blood (HLA-A*02:01, A*24:02) transplantation following myeloablative conditioning regimen in first complete molecular remission. We analyzed the cytomegalovirus-specific CD8(+) T cells (CMV-CTLs) through HLA-A*02:01 tetramer (HLA-mismatched) and HLA-A*24:02 tetramer (HLA-matched) by flow cytometer. T cell receptor repertoire of these tetramer-positive CMV-CTLs were analyzed by CTL cloning and CDR3 sequencing. [Results / Discussion]The patient had a short period of CMV antigenemia but not CMV disease which was well-controlled by drip infusion of the anti-viral reagent ganciclovir after transplantation. On day 41 after transplantation, only A24-restricted (HLA-matched) tetramer-positive CMV-CTLs were present. By day 195, however, A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs also appeared. We considered that the donor-derived dendritic cells (DCs) induced A2-restricted (HLA-mismatched) tetramer-positive CMV-CTLs from naïve T cells, which are originally contained in cord blood inoculum. These mismatched donor HLA restricted memory T cells existed for a long time with a wide-ranging repertoire after transplantation, however, they were considered to have a limited functional role in infection immunity because lungs and intestinal tracts epithelial cells do not express HLA-A2. Usually, cord blood with 0, 1, or 2 loci mismatches is applicable for transplantation. Since HLA-A and B loci are checked only on a serological level, there are some possibilities in which every HLA-A and B loci are mismatched in the genetic level. In such a case, induction of virus-specific CTLs from donor naïve T cells might be impaired and result in uncontrollable viral infections. The findings of this case will be helpful to understand the impact of HLA-mismatch on viral immunity. Disclosures:No relevant conflicts of interest to declare.

The Absence of CC-Chemokine Receptor 8 on Donor Regulatory T Cells Impairs Their Ability to Prevent Lethal Acute Gvhd.

AbstractAbstract 3743CC-Chemokine receptor 8 (CCR8) is a chemokine receptor expressed on the surfaces of activated effector T cells (Teffs) and resting regulatory T cells (Tregs). CCR8 has previously been shown to play a role in the trafficking of memory T cells into cutaneous and possibly pulmonary sites, and to contribute to the ability of Tregs to properly interact with antigen presenting cells within secondary lymphoid tissue. Since the lungs and in particular the skin are frequent sites of tissue injury in those developing graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), we set out to study the contribution of CCR8 to GVHD pathogenesis using a haploidentical murine HSCT model. Given the emerging utilization of therapeutic donor Treg infusions as a means for preventing GVHD, we also explored the role that CCR8 plays in the protective immunosuppressive function of Tregs in the HSCT setting. Methods:For our studies we used a haplotype-matched murine transplant model. C57BL/6 mice (B6; H-2b) functioned as donors, and B6 × DBA2 (B6D2; H-2bxd) mice served as recipients. For Teff studies, B6D2 mice were lethally irradiated to 950 rads on transplant day -1, and then administered 3×106 T cell depleted (TCD) B6 bone marrow (BM) cells +/− 4×106 CD25− Teffs isolated from the spleens of wild-type (WT) or CCR8−/− B6 mice on day 0. For studies involving regulatory T cells, B6D2 animals were lethally irradiated on day -1 and administered WT B6 TCD BM cells +/− 1–1.25×106 CD4+CD25+ Tregs isolated from WT or CCR8−/− B6 mice on day 0. 4×106 WT B6 Teffs were then administered on transplant day +2. For some transplants, WT B6 and CCR8−/− animals transgenic for enhanced green-fluorescent protein (eGFP+) were used as Teff and/or Treg donors in order to study T cell trafficking following HSCT. Both stereo-fluorescence microscopy and anti-eGFP ELISA approaches were utilized for these in vivo trafficking experiments. The in vitro suppressive potential of WT and CCR8−/− Tregs was evaluated by examining their ability to inhibit Teff proliferation in a one way mixed lymphocyte reaction (MLR). Results:CCR8 was not required for the induction of lethal GVHD, with recipients of CCR8−/− Teffs demonstrating a 100% mortality rate by transplant day +32. However, CCR8 was important for Treg function. In vivo, CCR8−/− Tregs were significantly less effective at preventing GVHD lethality than WT Tregs (see figure; P=0.013 for day +70 survival proportion comparison using Fisher's exact test). When we evaluated donor Treg trafficking on transplant days 6–7 using eGFP+ cells, no significant differences were noted between those animals receiving WT versus CCR8−/− Tregs. By transplant days 9–10, however, a generally higher eGFP signal was observed by stereo-fluorescence microscopy within the lymph nodes of those animals receiving WT eGFP+ Tregs compared to those given CCR8−/− eGFP+ Tregs. In addition, significantly more eGFP was detected by ELISA in the spleens and colons of WT eGFP+ Treg recipients (P=0.0015 and 0.021 respectively for mean total eGFP comparison between WT and CCR8−/− Treg groups by student's t test), and a strong trend for greater WT Treg accumulation was noted within host livers and lungs (P=0.059 and 0.066 respectively). Finally, CCR8−/− Tregs were relatively impaired in their ability to suppress T cell proliferation in vitro compared to WT cells, particularly at higher dilutions. [Display omitted] Conclusions:CCR8 is not required for donor Teffs to induce lethal GVHD in our HSCT model. However, CCR8 expression does appear to be important for the function of Tregsin vitro and in vivo, particularly at limiting Treg dilutions. CCR8 is not required for the accumulation of donor Tregs within any one recipient site. Rather, CCR8 appears to globally potentiate the accumulation of donor Tregs within most recipient tissues, with the extent of this effect increasing over time. Future experiments will focus on elucidating a mechanism for this finding. Disclosures:No relevant conflicts of interest to declare.

Recombinant Bactericidal Permeability Increasing Protein (rBPI 21) For Endotoxin (Lipopolysaccharide, LPS) Neutralization During Hematopoietic Stem Cell Transplantation (HSCT): Phase I/II Clinical Trial Results

Mucosal barrier injury caused by HSCT chemoradiotherapy conditioning leads to translocation of bacteria and bacterial products, including LPS-bearing bacteria and free LPS, into the systemic circulation. LPS signaling through its TLR4 receptor results in inflammatory cytokine release and resulting hypotension, fever, coagulopathy and end organ dysfunction. In allogeneic murine HSCT models, prior immunization to LPS, blockade of LPS:TLR4 signaling and use of LPS hyporesponsive mice all decrease HSCT morbidity and mortality, particularly acute GVHD and interstitial pneumonia.

Absence of β7 integrin results in less graft-versus-host disease because of decreased homing of alloreactive T cells to intestine

The alpha4beta7 integrin plays a central role in the homing of T cells to the gut. We hypothesized that absence of the beta7 subunit would result in a reduction of intestinal graft-versus-host disease (GVHD) and an improvement in overall GVHD morbidity and mortality in recipients of hematopoietic stem cell transplantation (HSCT). Analysis of alloreactive beta7-/- T cells showed intact activation, proliferation, cytokine production, and cytotoxicity. However, recipients of beta7-/- donor T cells in murine HSCT models experienced less GVHD morbidity and mortality than recipients of wild-type (WT) T cells, associated with a decrease in donor T-cell infiltration of the liver and intestine and with an overall significant decrease in hepatic and intestinal GVHD. In graft-versus-tumor (GVT) experiments, we demonstrated intact or even enhanced GVT activity of beta7-/- donor T cells. In conclusion, beta7-/- donor T cells caused less GVHD morbidity and mortality than WT donor T cells because of selectively decreased T-cell infiltration of the liver and intestines. Our data suggest that strategies to target the beta7 integrin have the clinical potential to alleviate or prevent GVHD while sparing or potentiating GVT activity.

Cotransplantation of Marrow Stromal Cells May Prevent Lethal Graft-versus-Host Disease in Major Histocompatibility Complex Mismatched Murine Hematopoietic Stem Cell Transplantation

Marrow stromal cells (MSC) produce a microenvironment supporting hematopoiesis and may contribute immune tolerance because of low immunogenicity and the suppressive effect of alloreactivity. We investigated whether cotransplantation of MSC could prevent lethal graft-versus-host disease (GVHD) in major histocompatibility complex mismatched allogeneic murine hematopoietic stem cell transplantation (HSCT) using female BALB/c (H-2d, recipient) and C3H/He (H-2k, donor) mice. MSC were obtained from C3H/He bone marrow cells (BMC). MSC and irradiated BALB/c splenocytes (SP) were cocultured with C3H/He SP or BMC. Nonirradiated MSC did not inhibit the proliferation of alloantigen-stimulated BMC and SP. However, irradiated MSC suppressed the proliferation of alloantigen-stimulated SP at a level comparable with that of immunosuppressive agents, and the suppression by MSC was reversed to a significant degree by interleukin 2. Lethally irradiated BALB/c mice received transplants of donor cells according to the following experimental groups (group A, BMC only; group B, BMC and SP; group C, BMC, SP, and MSC; group D, BMC and MSC). The survival rate in group D was higher than in the other groups (P = .0057), and the clinical GVHD scores and serum levels of interferon-gamma were low in group D. Our results suggest that cotransplantation of MSC in HSCT prevents lethal GVHD, possibly by immune modulation.

Effects of Cyclosporin A, FK506, and 3-Deazaadenosine on Acute Graft-versus-host Disease and Survival in Allogeneic Murine Hematopoietic Stem Cell Transplantation

Background: We investigated the effect of donor marrow T cell depletion, administration of FK506, cyclosporin A (CSA), and 3-deazaadenosine (DZA) on graft versus host disease (GVHD) after allogeneic murine hematopoietic stem cell transplantation (HSCT). Methods: We used 4 to 6 week old Balb/c (H-2, recipient), and C3H/He (H-2, donor) mice. Total body irradiated recipients received 1×10 bone marrow cells (BM) and 0.5×10 splenocytes of donor under FK506 (36 mg/kg/day), CSA (5 mg/kg/day, 20 mg/kg/day), and DZA (45 mg/kg/day), which were injected intraperitoneally from day 1 to day 14 daily and then three times a week for another 2 weeks. To prevent the GVHD, irradiated Balb/c mice were transplanted with 1×10 rotor-off (R/O) cells of donor BM. The severity of GVHD was assessed daily by clinical scoring method. Results: All experimental groups were well grafted after HSCT. Mice in experimental group showed higher GVHD score and more rapid progression of GVHD than the mice with R/O cells (R/O group) (p<0.01). There were relatively low GVHD scores and slow progressions in FK506 and low dose CSAgroups than high dose CSA group (p<0.01). The survival was better in FK506 group than low dose CSA group. All mice treated with CSA died within 12 days after HSCT. The GVHD score in DZA group was low and slow in comparison with control group (p<0.05), but severity and progression were similar with low dose CSA group (p=0.11). All mice without immunosuppressive treatment died within 8 days, but all survived in R/O group (p< 0.01). Survival in low dose CSA group was longer than in control group (p<0.05), but in high dose CSA group, survival was similar to control group. The survival benefit in DZA group was similar with low dose CSA group. FK506 group has the best survival benefit than other groups (p<0.01), comparable with R/O group (p=0.18), although probability of survival was 60%. Conclusion: We developed lethal GVHD model after Effects of Cyclosporin A, FK506, 3-Deazaadenosine on Acute GVHD 151

Inhibition of Graft Versus Host Disease Using CD4+ CD25+ T Cells Induced with Interleukin-2 in Mismatched Allogeneic Murine Hematopoietic Stem Cell Transplantation

Background: In kidney transplantation, donor specific transfusion may induce tolerance as a result of some immune regulatory cells against the graft. In organ transplantation, the immune state arises from a relationship between the immunocompromised graft and the immunocompetent host. However, a reverse immunological situation exists between the graft and the host in hematopoietic stem cell transplantation (HSCT). In addition, early IL-2 injections after an allogeneic murine HSCT have been shown to prevent lethal graft versus host disease (GVHD) due to CD4+ cells. We investigated the induction of the regulatory CD4+CD25+ cells after a transfusion of irradiated recipient cells with IL-2 into a donor. Methods: The splenocytes (SP) were obtained from 6 week-old BALB/c mice (H-2) and irradiated as a single cell suspension. The donor mice (C3H/ He, H-2) received 5×10 irradiated SP, and 5,000 IU IL-2 injected intraperitoneally on the day prior to HSCT. The CD4+CD25+ cell populations in SP treated C3H/He were analyzed. In order to determine the in vivo effect of CD4+CD25+ cells, the lethally irradiated BALB/c were transplanted with 1×10 donor BM and 5×10 CD4+CD25+ cells. The other recipient mice received either 1×10 donor BM with 5×10 CD4+ CD25cells or the untreated SP. The survival and GVHD was assessed daily by a clinical scoring system. Results: In the MLR assay, BALB/c SP was used as a stimulator with C3H/He SP, as a responder, with or without treatment. The inhibition of proliferation was 30.0±13% compared to the control. In addition, the MLR with either the CD4+CD25+ or CD4+CD25cells, which were isolated by MidiMacs, from the C3H/He SP treated with the recipient SP and IL-2 was evaluated. The donor SP treated with the recipient cells and IL-2 contained more CD4+CD25+ cells (5.4±1.5%) than the untreated mice SP (1.4±0.3%)(P<0.01). There was a profound inhibition in the CD4+CD25+ cells (61.1±6.1%), but a marked proliferation in the CD4+CD25cells (129.8±65.2%). Mice in the CD4+CD25+ group showed low GVHD scores and a slow progression from the post-HSCT day 4 to day 9, but those in the control and 287 Immune Network ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ 책임저자:정대철, 성모자애병원 소아과, ꂕ 403-720, 인천시 부평구 부평 6동 665 Tel: 032-510-5687, Fax: 032-503-9724, E-mail: dcjeong@olmh.cuk.ac.kr 이 논문은 2003년 가톨릭중앙의료원 성의장학 연구비원과제비에 의해 이루어졌음. 288 Jae Ho Hyun, et al

Myeloid progenitors protect against invasive aspergillosis andPseudomonas aeruginosa infection following hematopoietic stem cell transplantation

Myelotoxic treatments for oncologic diseases are often complicated by neutropenia, which renders patients susceptible to potentially lethal infections. In these studies of murine hematopoietic stem cell transplantation (HSCT), cotransplantation of lineage-restricted progenitors known as common myeloid progenitors (CMP) and granulocyte-monocyte progenitors (GMP) protects against death following otherwise lethal challenge with either of 2 pathogens associated with neutropenia: Aspergillus fumigatus and Pseudomonas aeruginosa. Cotransplantation of CMP/GMP resulted in a significant and rapid increase in the absolute number of myeloid cells in the spleen, most of which were derived from the donor CMP/GMP. Despite persistent peripheral neutropenia, improved survival correlated with the measurable appearance of progenitor-derived myeloid cells in the spleen. A marked reduction or elimination of tissue pathogen load was confirmed by culture and correlated with survival. Localization of infection by P aeruginosa and extent of disease was also assessed by in vivo bioluminescent imaging using a strain of P aeruginosa engineered to constitutively express a bacterial luciferase. Imaging confirmed that transplantation with a graft containing hematopoietic stem cells and CMP/GMP reduced the bacterial load as early as 18 hours after infection. These results demonstrate that enhanced reconstitution of a tissue myeloid pool offers protection against lethal challenge with serious fungal and bacterial pathogens.

In vivo administration of granulocyte colony-stimulating factor (G- CSF), granulocyte-macrophage CSF, interleukin-1 (IL-1), and IL-4, alone and in combination, after allogeneic murine hematopoietic stem cell transplantation

BALB/c mice (H-2d) given 10 Gy total body irradiation (TBI) followed by 10(7) bone marrow (BM) and 10(6) spleen cells from C57BL/6 (H-2b) donor mice received recombinant cytokines intraperitoneally (IP) twice daily. The effect on neutrophil recovery rate, graft-v-host disease (GVHD), and survival was assessed. Four reagents were used: granulocyte-colony- stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin-1 (IL-1) and IL-4, both alone and in combination. The most effective combination for increasing the circulating absolute neutrophil account (ANC) above the control value at day 7 posttransplant was the combination of G-CSF and IL-1 (mean ANC 2.4 +/- 1.6 x 10(9)/L as compared with control value of 0.07 +/- 0.05, P less than .02), followed by G-CSF alone (mean ANC 1.1 +/- 0.2, P less than .0001), the combination of GM-CSF plus IL-1 (mean ANC 0.8 +/- 0.3, P less than .002), and the combination of G-CSF plus GM-CSF (mean ANC 0.8 +/- 0.3, p less than .005). At day 10 posttransplant, the most effective combination in raising the ANC was the combination of G-CSF plus GM-CSF (mean ANC 7.5 +/- 2.3 as compared with control value of 3.5 +/- 1.1, P less than .01), followed by G-CSF alone (mean ANC 6.9 +/- 2.1, P less than .02). At the doses used, neither G-CSF nor GM-CSF had a deleterious effect on the incidence or severity of GVHD; indeed, GM- CSF was associated with improved survival. In contrast, IL-1 at doses greater than or equal to 100 ng twice daily caused marked early mortality, and there was a suggestion that IL-4 at doses of 500 ng twice daily resulted in increased late mortality, possibly owing to exacerbation of GVHD. This model appears to be of value for exploring the use of hematopoietic growth factors before they are used clinically in marrow allograft recipients.